The study of cardiac myosin binding protein C and associated mutations in the MYBPC3 gene has provided a more comprehensive understanding of hypertrophic cardiomyopathy in humans and domestic felines. Our knowledge about this disease as a whole, however, remains quite limited, necessitating investment in further research. Additional research should focus on identifying the complete spectrum of polymorphisms in the MYBPC3 gene that do not cause HCM for genetic comparison with mutations in these allelic variants. Elucidating the specific functions of cMyBP- C is also fundamental to the appreciation of the molecular basis for HCM. Furthermore, continued study of the naturally occurring animal model for the disease, the domestic cat, may also prove essential to gaining a complete understanding of disease pathogenesis and the development of prospective treatments. Finally, with continued research, more knowledge can be gained about normal cardiac function. This, in turn, could be applied to understanding cardiovascular disease to develop alternative treatments for these devastating diseases.
Hypertrophic cardiomyopathy (HCM), characterized by left ventricular hypertrophy and fibrosis, is the most common monogenetic form of inherited heart disease affecting approximately 1 in 500 individuals. Patients with HCM are predisposed to heart failure, outflow tract obstruction, arrhythmias, and sudden cardiac death. The most frequently mutated gene in HCM is cardiac myosin binding protein C (MYB- PC3), accounting for more than 50% of cases in which the causative gene has been identified (1). MYB- PC3 is a 150-kDa myofilament protein found in the C-zone of the thick filament that interacts with both myosin and actin, acting as a molecular brake on cross-bridge cycling (2). In contrast to other sarcomere genes that primarily harbor missense mutations, over 90% of MYBPC3 mutations are nonsense, result- ing in premature termination and predicted to yield truncated proteins (1, 3). Yet, multiple studies have failed to identify the presence of mutant truncated MYBPC3 in myocardium from HCM patients (4–6). Nonsense mutant MYBPC3 transcript has been shown to undergo nonsense-mediated decay (NMD), suggesting the majority of mutant transcript is degraded (7). However, we have previously shown that in myocardium from patients with MYBPC3-linked HCM, transcript originating from the mutant allele of MYBPC3 is still detectable in sufficient quantities to provide a template for mutant protein synthesis (8). While the absence of truncated proteins supports a disease mechanism arising from haploinsufficiency of full-length MYBPC3 within the sarcomere, a contribution from mutant protein expression has not been conclusively excluded. In several studies using both cell and animal models, expression of truncat- ed mutant MYBPC3 protein was associated with impairment of the ubiquitin-proteasome system (UPS), one of the major cellular protein degradation pathways (9–11). Deficits in the UPS have been previously linked to human HCM particularly when caused by truncating mutations in MYBPC3 (12, 13). UPS dysfunction is an indicator that protein homeostasis, or proteostasis, is disrupted. However, what is not
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Analysis of LV-chamber systolic and diastolic prop- erties was performed in vivo as described (31). In brief, a miniaturized impedance/micromanometer catheter was used to derive real-time LV pressure-volume rela- tionships. Animals were anesthetized with etomidate (10–20 mg/kg body weight), morphine (1–2 mg/kg body weight), and urethane (750 mg/kg body weight), intubated, and artificially ventilated with a custom- designed murine ventilator, with 100% inspired oxygen at a tidal volume of 200 µL and frequency of 120 breaths per minute. Heart rates were near normal (>500 beats per minute). The LV was catheterized using an apical stab exposed by a limited thoracotomy. Signals were recorded at steady state and during tran- sient load reduction produced by transiently occlud- ing the inferior vena cava. Data were sampled at 2 kHz and analyzed with custom software. Following initial data collection, an ultrasound perivascular flow probe (1RB; Transonics, Ithaca, New York, USA) was placed around the thoracic aorta to measure cardiac output. The stroke volume was used to calibrate relative vol- umes measured simultaneously by the volume- catheter signal, matching its value to the mean width of the pressure-volume loop. Relative changes and the majority of derived hemodynamic parameters were cal- ibrated, although absolute volume was not. Heart weights and gross anatomic examinations were made after completion of each study. Comparisons between wild-type and mutant MyBP-C mice were performed by Student t tests. All data are reported as means ± SEM and P values less than 0.05 are numerically reported; higher values are indicated as nonsignificant.
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Two previous studies have described the effects of COOH- terminally truncated MyBP-C transfected into vertebrate myo- cytes (32, 43). These experiments, especially the detailed study by Gilbert et al. (32), highlighted the complex interactions in the COOH terminus of MyBP-C involved in sarcomere assem- bly. It was demonstrated that the COOH-terminal Ig domain is essential for thick-filament targeting. Domains C8–C10 are minimal requirements for A-band incorporation. The lack of domains C10 and/or C9 causes myofibrillar disarray only in the presence of domain C8, while these constructs are not incorpo- rated into the A-band in detectable amounts. In contrast, COOH-terminally truncated constructs shorter than domain C8 were neither dominant negative nor localized to the A-band. Additional protein interactions, localized in the region that binds to titin in vitro (27, 32), appear to be crucial for correct sarcomeric assembly. The observed myofibrillar disarray thus seems to be caused by competition of truncated MyBP-C with A-band components, most likely titin. However, the transfec- tion studies in cell culture with forced overexpression of mu- tated protein under control of strong viral promoters resulted in the rapid disturbance of sarcomere assembly (32, 43), which contrasts with the late manifestation of FHC in family mem- bers carrying the mutation and with the rather slow progres- sion of the disease in clinically affected patients. Furthermore, the splice acceptor mutation of MyBP-C described by Bonne et al. (8) is predicted to produce a truncated protein, which is lacking the domains C5–C10. The transfection studies by Gil- bert et al. (32) did not reveal any significant dominant negative effect on myofibrillar assembly for the similar construct D7-10. Therefore, cell culture models and transfection assays may not reflect the assembly control of myofibrillar proteins in the dis- eased myocardium and therefore the pathogenesis of chromo- some-11–associated FHC.
Dilated cardiomyopathy (DCM) is the most common type of cardiomyopathies seen clinically. Its pathogenesis is not yet fully understood. Its occurrence and development may be attributed to secondary causes such as viral infection, hypertension, autoimmune cause, alcohol intake and drugs. It has been reported that the 5-year survival rate of DCM is less than 50% after the emergence of DCM symptoms  ; in China, the prevalence rate is 19 per 100,000 . Pa- thologically, DCM changes are characterized by a triad of myocyte degradation, myocyte hypertrophy and myocardial fibrosis . DCM can also be due to idi- opathic causes, which is principally due to gene mutations in specific cardiac proteins  . The main clinical manifestations of DCM patients are chest tightness, palpitation, dyspnea and decreased activity tolerance, serious symp- toms of heart failure in more severe cases, and even appearance of serious heart arrhythmias. Transplantation is the only effective treatment. Current treatment methods are only to improve the symptoms of heart failure and to prevent com- plications. DCM mainly manifests as a declination of systolic and diastolic func- tions. There are a wide variety of structural cardiac proteins involved in myocar- dial contraction and relaxation. One protein of great interest is cMyBP-C. The latter is an important regulotory component of the sarcomere which ensures proper regulation of heart systolic and diastolic function. In this review, we em- phasize on an update about the research progress of cMyBP-C and its relation to DCM and other cardiac conditions.
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hood, mirroring the relatively benign clinical picture of the MyBP-C–linked FHCs. While analyses at the whole organ level were uninformative, at the skinned fiber level, using very young animals in which cardiomyocyte degeneration had not yet pre- sented, significant changes in the maximum relative power and the sensitivity of force production to Ca 21 levels could be dis- cerned, indicating that subtle alterations in force production oc- cur. Although there is no apparent heart enlargement and myo- cardium wall thickening in the MyBP-C. mut1 hearts, to date we have not yet obtained a large, aged cohort of animals suit- able for detailed morphometric and stress exercise analyses (36). Previously, in other mouse models we have detected very early signs of hypertrophy using RNA analyses (37). However, molecular markers of hypertrophy such as b -MyHC, a -skeletal actin, and atrial natriuretic factor were not present in RNA de- rived from 6-mo MyBP-C.mut1 line 32 animals (data not shown). We suspect that the hypertrophic process in these mice is very slow or minimal, despite the scattering of dysfunctional cells in the myocardium. Consistent with this hypothesis, immu- nostaining and ultrastructural analyses of a very limited num- ber of older ( . 30 wk) animals showed a high degree of myocyte disarray and the nuclei, as visualized by electron mi- croscopy, displayed the highly convoluted periphery character- istic of cardiomyocytes undergoing hypertrophy (data not shown). These data are particularly intriguing in light of clinical data that show onset of MyBP-C–FHC occurs much later in life than the FHCs caused by mutations in other contractile pro- teins (38). Again, it will be necessary to study a large, aged co- hort of animals in order to define these processes completely.
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Genetic mutations account for a significant percentage of cardiomyopathies, which are a leading cause of conges- tive heart failure. In hypertrophic cardiomyopathy (HCM), cardiac output is limited by the thickened myocardium through impaired filling and outflow. Mutations in the genes encoding the thick filament components myosin heavy chain and myosin binding protein C (MYH7 and MYBPC3) together explain 75% of inherited HCMs, leading to the observation that HCM is a disease of the sarcomere. Many mutations are “private” or rare variants, often unique to families. In contrast, dilated cardiomyopathy (DCM) is far more genetically heterogeneous, with muta- tions in genes encoding cytoskeletal, nucleoskeletal, mitochondrial, and calcium-handling proteins. DCM is charac- terized by enlarged ventricular dimensions and impaired systolic and diastolic function. Private mutations account for most DCMs, with few hotspots or recurring mutations. More than 50 single genes are linked to inherited DCM, including many genes that also link to HCM. Relatively few clinical clues guide the diagnosis of inherited DCM, but emerging evidence supports the use of genetic testing to identify those patients at risk for faster disease progression, congestive heart failure, and arrhythmia.
tissue culture flask were lysed using 200 l of cell lysis buffer (20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 0.05% sodium dodecyl sulfate [SDS], 5 mg/ml sodium deoxycholate, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluo- ride) at 4°C for 10 min. Cell genomic DNA was pelleted by centrifugation at 13,000 ⫻ g for 10 min at 4°C. Cell lysate supernatant was either stored at ⫺80°C or used for protein analysis. Approximately 100 g of total protein was run on a 10% SDS-polyacrylamide gel, and proteins were subsequently transferred to a nitrocellulose membrane (Pall, Pensacola, FL). V5-tagged FLVCR1/FLVCR2 proteins were detected by incubation of nitrocellulose membranes with anti-V5 monoclonal antibody (Invitrogen) diluted 1 in 500 in phosphate-buffered saline (PBS) containing 0.1% Tween 20. This was followed by incubation with rabbit anti-mouse antibody conjugated to horseradish peroxidase (HRP) (Sigma) di- luted 1 in 1,000 in PBS–0.1% Tween 20. HA-tagged proteins were detected by incubation of nitrocellulose membranes with the anti-HA HRP antibody (Sigma- Aldrich). Signals were detected using chemiluminescence reagent (Perkin Elmer, Boston, MA), followed by exposure to Kodak Biomax MR film. For loading control of cell lysate samples, the nitrocellulose membrane was incubated with anti-actin monoclonal antibody (diluted 1 in 1,000; Sigma-Aldrich) followed by goat anti-mouse HRP (diluted 1 in 1,000; Sigma-Aldrich). Cell membrane sam- ples were prepared from cells grown to confluence in 150-mm diameter tissue culture plates. Cells were initially washed with PBS and then resuspended in 3 ml of cold membrane lysis buffer (20 mM Tris [pH 7.4], 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 mM aprotinin). The cells were scraped from the tissue culture dish using a cell scraper and then homogenized using a Dounce homogenizer. The nuclear fraction was pelleted by centrifugation at 1,000 ⫻ g for 20 min at 4°C. Membrane fractions were pelleted by centrifugation of the nu- cleus-free supernatant at 30,000 rpm for 1 h at 4°C in a Beckman SW41 rotor. The membrane pellet was resuspended in 40 l of PBS. Twenty microliters of membrane sample was run on a 10% SDS-polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane, and V5- and HA-tagged receptor proteins were detected as described above. For loading control, the remaining 20 l of membrane sample was run on another 10% gel and transferred to a nitrocellulose membrane, which was subsequently incubated with a monoclonal antibody against the ␣ subunit of the sodium potassium ATPase membrane protein (Sigma-Aldrich).
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Washed beef meat (WBM) is surimi-like product made from red beef meat. The process for making surimi-like product from beef, using modified technology from fish surimi (Park et al. 1996), results in a semi-purified protein fraction contain- ing a high concentration of myofibrillar proteins. Freezing has become one of the most frequently used preservation methods for meat and meat products. To protect myofibrillar proteins from freeze-denaturation and during frozen storage and to maintain its possible high processability, cryoprotectants, such as disaccharides, polysac- charides, polyalcohols, acids, or polyphosphates are generally added (Park et al. 1988; Mac Donald & Lanier 1991). The most commonly used in- strumental methods for the determination of the cryoprotective effects of the added substances are the measurement of myofibrillar protein solubility SEP (Salt extractable protein) (Sych et al. 1990),
was visualized directly (Fig. 7). RNAP II was seen in globular replication compartments in KOS- and vN-YFP-ICP27-in- fected cells, but a more diffuse nuclear fluorescence was seen for RNAP II in vNC-Venus-ICP27-infected cells (Fig. 7). This pattern resembled that of the cell that did not show Venus fluorescence in the nNC-Venus-ICP27 panels and that may represent an uninfected cell (Fig. 7). Further, while ICP4- containing viral replication compartments were seen in KOS- and vN-YFP-ICP27-infected cells, smaller prereplication sites were seen in vNC-Venus-ICP27-infected cells (Fig. 7). Thus, FIG. 6. Recombinant virus v-NC-Venus-ICP27 is defective in viral replication. The v-NC-Venus-ICP27 virus was constructed by the marker transfer of N-Venus/ICP27/C-Venus into 27-LacZ (40), replacing the 27-LacZ locus. (A) One-step growth curves were performed by infecting Vero cells with KOS, 27-GFP, and vNC-Venus-ICP27 at an MOI of 1. The experiments were performed in triplicate, and viral titers at 4, 8, 16, and 24 h were determined by plaque assays on Vero cells. (B) Vero cells were infected with v-NC-Venus-ICP27 and v-N-YFP-ICP27 at an MOI of 10. Venus and YFP fluorescence were visualized directly on living cells at 4, 6, and 8 h after infection. (C) Vero cells were infected with KOS, v-N-YFP-ICP27, and v-NC-Venus-ICP27 at an MOI of 10. Western blot analysis was performed on protein samples isolated at 4, 8, and 12 h after infection. The blot was probed with anti-ICP27 and anti-GFP antibodies and with ␤ -actin antibody as a loading control. The red arrows indicate the position of wild-type ICP27. The green arrows indicate the position of N-YFP-ICP27 and N-Venus/ICP27/C-Venus. The asterisks denote ICP27 protein degradation products in the N-YFP-ICP27 lanes. The position of molecular size markers is shown to the left of the gel. (D) Vero cells infected with KOS, vN-YFP-ICP27, and v-NV-Venus-ICP27 at an MOI of 10 were harvested at 4, 8, and 12 h after infection, and Western blot analysis was performed as described previously (42). The blots were probed with antibodies directed to ICP4, gC, gD, and ␤ -actin as a loading control.
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The sepsis cascade, which is associated with simultaneous activation of systemic inflammation and coagulation, along with altered fibrinolysis, leads to microvascular endothelial injury, acute organ dysfunction, and possibly death. Throm- bin, with its procoagulant and anticoagulant effects, as well as its involvement in the process of inflammation, is thought to be central to the sepsis cascade. In vitro and animal models suggest that Activated Protein C, a protein that has antithrombotic, anti-inflammatory and profibrinolytic activi- ties, may be an important modulator of the vicious cycle of coagulation and inflammation associated with the sepsis cascade, which if unchecked may ultimately lead to death. Several properties of Activated Protein C, including its unique positioning to regulate coagulopathies in the microvasculature and the ability of EPCR to facilitate Protein C activation, support the continued development of Acti- vated Protein C-mediated strategies that are aimed at dis- rupting the process whereby inflammation initiates coagulation and coagulation amplifies inflammation.
A conserved glycine sepaiating the two short helices caiiying the reactive thiol groups (SH I and SH2) is essential for motor activity and may be the pivot point for the lever aim (Kinose eta l., 1996). Lying under the nucleotide binding pocket, the SHI helix is dkectly linked to the regulatory domain and interacts with a small helix of highly conserved residues which originates at the base of the cleft neai* the putative y-phosphate binding site. This suggests that the thiol containing helix may play a major role in the transduction of the small sti'uctural changes occum ng in the head domain to the regulatoiy domain where they are amplified into movement. Exactly how this is amplified by the light chain binding domain is unknown. A ltering the structure of the regulatoi'y domain affects filament velocity and force production but has little effect on ATPase activity. Myosin with the regulatory domain rem oved show a large decrease in filam ent velocity (Uyeda and Spudich, 1993). Shortening or lengthening the regulatory domain by the removal or addition of one light chain binding site results in a lineai' reduction or increase in filament velocity respectively (Uyeda et al., 1996). Recent work suggests striking similarities between the structure and mechanisms utilised by myosin (and kinesin) and G proteins (Vale, 1996). Such com paiisons should help us understand further the mechanisms involved in nucleotide hydrolysis and energy transduction and how this effects myosin function.
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cellular membrane microenvironments. The three WNV chimeras that displayed both low levels of early viral RNA synthesis and SG induction similar to Eg101 were Eg-NS5, Eg-NS1+3+4a and Eg-NS1+5. The polyprotein sequences of Eg101 and W956IC were aligned and revealed a number of amino acid changes in the nonstructural proteins as there are 32 aa changes in NS1, 30 aa changes in NS3+4a, and 33 aa changes in NS5. The majority of these mutations are within the same class and are unlikely to modify the function of these proteins. These analyses identified five mutations within NS1, NS3+4a and NS5 that are unique to W956IC and D117B956 compared to the various WNV strains tested and are currently being tested for their effect on early viral replication efficiency and SG induction. However, it is more likely that the difference in replication efficiencies between these viruses is due to a combination of mutations and not to a sole residue mutation as evidenced by the lack of a single viral protein causing the difference in SG induction. The finding that chimeras with either an NS5 with both its N- and C-terminal regions from Eg101 or a combination of the Eg101 NS1, NS3 and NS4a proteins produced low levels of early viral RNA synthesis support the hypothesis that viral nonstructural protein conformations and interactions within replication complexes play a role in down-regulating RNA synthesis early during natural WNV infections. Similarly, a previous study mapping flavivirus nonstructural proteins involved in inhibiting IFN signaling also did not identify a single protein as the cause for the difference and further supports the findings of the present study (Ambrose and Mackenzie, 2011).
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in plasma samples by commercially available enzyme- linked immunosorbent assays (ELISA). Plasma sCD14 was quantified using the Quantikine Human sCD14 Immunoassay (R&D Systems, Minneapolis, MN), and plasma LBP was measured by LBP soluble ELISA kit (Hycult Biotechnology, Uden, The Netherlands) according to the manufacturers’ protocols. Plasma bacterial endo- toxin, i.e., LPS, was measured from heparinized blood samples (Brandtzaeg) using the Limulus Amebocyte Lysate (LAL) assay (Lonza Group Ltd, Allendale, NJ). The method uses a chromogenic endpoint assay yielding data as endo- toxin units (EU/ml). Briefly, 100 μl of each plasma sample was diluted in 200 μl of β-G-Blocker (Lonza Group Ltd, Allendale, NJ) to eliminate the possibility of false positives. Samples were further diluted with 100 μl of pyrogen-free water to give a final dilution of 1:4. All dilutions were pre- pared in pyrogen-free tubes. Samples were then placed in a water bath at 85 °C for 15 min to inactivate inhibitory plasma proteins. Results of LPS measured were expressed in picograms per milliliter (1 EU/ml = 100 pg/ml). Levels of intestinal fatty acid binding protein (I-FABP), a marker associated with enterocyte damage, were assayed using an ELISA (Hycult Biotechnology, Uden, the Netherlands) ac- cording to the manufacturer’ s instructions. All the samples were run in duplicate.
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Lymphocytes binding C-reactive protein (CRP) were studied in 31 patients with acute rheumatic fever and 30 controls who were children. Marked elevations in both proportions and absolute numbers of CRP-binding lymphocytes were recorded in rheumatic fever (P less than 0.001). No clear correlation was noted between plasma CRP as quantitated by radioimmunoassay and proportions or numbers of CRP-binding cells. Double-labeling experiments indicated that 60-80% of CRP-binding lymphocytes also showed Fc receptors reacting with fluorescein-conjugated IgG aggregates. Passage of lymphocytes over Ig--anti- IgG columns, removed cells bearing surface Ig but not CRP-binding lymphocytes. Studies of T-cell subpopulations indicated no overlap between Tmicron- and CRP-binding cells;
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Total RNAs were prepared from C2C12 and myoblast cells by TRIzol (Life Technologies). RNA samples were reverse transcribed using random hexamer and oligo dT mixed primers with SuperScriptII enzyme (Life Techono- logies) according to the manufacturer’s instructions. Re- verse transcription reactions were diluted (1:10) with 10 mM Tris, pH 8.0, yielding master samples of reverse- transcribed products. Real-time PCR reactions are previ- ously described . Real-time data were gathered using a system (MX4000; Agilent Technologies, Santa Clara, CA, USA) over 40 cycles (30 s at 90°C, 60 s at 58°C and 30 s at 72°C) followed by a denaturation curve from 54°C to 94°C in 30-s increments of 0.5°C to ensure amplification specifi- city. Threshold cycle (Ct) values were calculated with the MX4000 software (Agilent Technologies, Santa Clara, CA, USA) by using moving window aver-aging and an adap- tive baseline. Fold changes, other calculations and chart plotting were performed in Microsoft Excel (Redmond, WA, USA). The sequence of PCR primers is listed in Additional file 1: Table S1.
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permeability to mannitol and inulin. After injury, cells shouldering wounds migrated, by extension of lamellipodia-like processes, to reseal wounds as defined by structural and functional criteria. F actin arcs crossed the base of the lamellipodia-like extensions and F actin microspikes projected from the leading edge of these extensions. Villin, an epithelial- specific cytoskeletal protein with both F actin bundling and severing capacities, was also expressed at the leading edge in a pattern consistent with a regulatory role in the dynamic restructuring of lamellipodia. Lastly, myosin II was predominantly localized to the basal regions of lamellipodia, though occasional staining was seen close to the advancing edge. Myosin I, a recently recognized myosin family member considered to be essential for fibroblast and slime mold motility, was present throughout lamellipodia in punctate fashion, but was not concentrated at the leading edge.
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Physical properties of actomyosin from either fresh or stored platelets have been compared. Actomyosin obtained from platelets after 3 days of storage contained myosin that was 60- 80% degraded to myosin rod. No myosin rod was detected in fresh platelets. The platelet myosin rod is similar to the rod produced by limited proteolysis of skeletal muscle myosin.
Plasmids. The plasmids pJFH1, pSGR-JFH1, and pSGR-Luc-JFH1 were the generous gift of T. Wakita (22, 48). pSGR-JFH1-5A1ST, in which NS5A gene contains the One-STrEP tag (IBA, Go ¨ttingen, Germany), was generated by PCR-mediated mutagenesis using oligonucleotides described in Table S1 in the supplemental material. pFL-Luc-Jc1, an analogue to Luc-Jc1 (23), was con- structed as described in Table S1 in the supplemental material. For constructing the human OSBP expression vector pFLAG-CMV-OSBP (where CMV is cyto- megalovirus), OSBP1 cDNA was amplified from total RNA extracted from Huh7 cells by reverse transcription-PCR (RT-PCR), using oligonucleotides described in Table S1 in the supplemental material, and amplified product was digested with both HindIII and BamHI and cloned into the corresponding sites in the pFLAG-CMV2 vector (Sigma-Aldrich, St. Louis, MO). Expression vectors for mutant forms of OSBPs were constructed as described in Table S1 in the supplemental material. The VAP-A expression vector, pEF-FLAG-VAP-A, was kindly provided by Y. Matsuura (17). Generation of NS5A deletion mutants has been described previously (20). The NS5A derived from HCV JFH1 (genotype 2a) was cloned in the pEF1/Myc-His vector (Invitrogen, Carlsbad, CA) at the KpnI and XbaI sites by using a PCR-amplified fragment using primers described in Table S1 in the supplemental material. Lentiviral vectors, L-CMV-GFP-NheI (where GFP is green fluorescent protein), and packaging plasmids pMDL, pVSV-G (where VSV-G is vesicular stomatitis virus glycoprotein), and pREV were kindly provided by I. Verma (Salk Institute, La Jolla, CA) (46) and used for cloning short hairpin RNAs (shRNAs). The oligonucleotides used for construct- ing a scrambled shRNA (unrelated) and the OSBP-specific shRNA-1 and shRNA-2 are described in Table S1 in the supplemental material.
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(diameter: 25 mm) before seeding (see subsection 3.5.1 for cleaning protocol). If live imaging was intended, cells were grown in glass bottom cell culture dishes. Transfection of plasmid DNA was implemented using Lipofectamine® 2000 Transfection Reagent (Thermo Fisher Scientific) following manufacturer´s instructions. Cells were incubated with Lipofectamine® 2000-DNA mixture for 4 h to 6 h. Afterwards, cells were left to grow for 24 h, 48 h, or 72 h before further treatment. Plasmids introduced into the cells contained a fluorophore sequence and DNA encoding a myosin-XXI construct. Furthermore, plasmids only encoding a fluorophore were used for control experiments. A detailed list of all plasmids used in transfections can be found in Table A.2. In addition to transfections with only one plasmid, mammalian cells were also co-transfected with either two plasmids or a plasmid and a baculovirus at a time. In co-transfection experiments, a plasmid containing myosin-XXI DNA and a construct for the staining of a certain cell structure were applied in order to acquire information about myosin-XXI cellular localization. The additional plasmid applied in co-transfections with two plasmids encoded a fusion protein of RFP (red fluorescent protein) and lifeact, a peptide used for staining of the actin cytoskeleton (Riedl et al., 2008). Baculoviruses (Cell Light® Reagents BacMam 2.0, Thermo Fisher Scientific) used in co-transfections encoded markers for early endosomes, Golgi apparatus, and endoplasmic reticulum. They were added to transfected cells immediately after cells had been incubated with Lipofectamine® 2000-DNA mixture. Baculovirus concentrations used varied between 1.2·10 6 and 1.6·10 6 particles per 10 5 cells. In all transfections and co-transfections performed, plasmid DNA was applied at a concentration of 0.2 µg to 2.2 µg per 10 5 cells and DNA to Lipofectamine® 2000 ratios ranged between 1:1.3 μg/μl and 1:4.6 μg/μl.
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