Deciliation of the trachea

The tracheas harvested from postnatal day 16 (P16) Dyx1c1 wildtype and knockout mice were washed with cold phosphate-buffered saline to remove mucus and cell debris. Animal procedures were performed under protocols approved by the Institutional Animal Care and Use Committee of the University of

Figure 5-5: Proposed role of Dyx1c1 in the cytoplasmic assembly pathway.

Dyx1c1 interacts with the chaperones and intermediate as well as light chain dynein subunits in the cytoplasm. The heavy chains are added onto the intermediate chains and the light chains are further incorporated into the outer dynein arm complex in the presence of Dyx1c1 and other assembly factors.

transferred to a separate tube containing deciliation buffer, and incubated for 10 min with intermittent mild vortexing. The supernatant was collected in a separate tube, and the procedure was repeated twice. The tracheas were then separated from the supernatant, the three washings were pooled, and after pelleting cell debris at 800xg, the ciliary axonemes were collected by centrifugation at 20,000xg. Ciliary axonemal pellets were resuspended gently in 30 mM Hepes (pH 7.3), 1 mM EGTA, 0.1 mM EDTA, 25 mM NaCl, 5 mM MgSO4, 1 mM dithiothreitol, and 1% volume of protease inhibitor mixture (Sigma). Triton X-100 was added (0.5%) to the suspension, and the axonemes were incubated for 15 min on ice, followed by centrifugation. The lysates containing the axonemal fraction were then frozen at -80°C. The cilia-stripped-tracheas were incubated in RIPA buffer (Sigma) on ice for 20 min. Samples were homogenized using a tissue homogenizer and cleared by centrifugation at 10,000g for 10 min to obtain the protein lysate of the cytoplasmic fraction of the trachea. Protein concentrations were estimated using the BCA reagent (Pierce).

5.5.2 Protein blots of whole trachea, cytoplasmic and axonemal lysates

For whole tracheal preparation, tracheal tissues were dissected from wildtype and knockout mice, and carefully separated from the surrounding tissues. After incubation in RIPA Buffer supplemented with 1× Protease Inhibitor Cocktail (Sigma), samples were homogenized using a tissue homogenizer and cleared by centrifugation at 10,000g for 10 min. The cytoplasmic and axonemal fractions of the trachea were prepared as described above. Proteins were separated on 12% SDS- PAGE minigels and then transferred to Immobilon (Millipore) membrane for

protein blotting. For detecting DYX1C1 protein, the antibody to N-terminal DYX1C1 (Sigma, SAB4200128) was used at a dilution of 1:200; antibody to acetylated tubulin (Sigma, T6793) was used at a dilution of 1:2000; and antibody to GAPDH (Sigma, G8795) was used at a 1:1,500 dilution as a loading control. LI-COR Odyssey infrared secondary antibodies (goat antibody to mouse 680 (926-32220), goat antibody to mouse 800 (926-32210), goat antibody to rabbit 680 (926-32221) and goat antibody to rabbit 800 (926-32211)) were used at dilutions of 1:15,000. All blots were imaged and analyzed using a LI-COR Odyssey Scanner and Software.

5.5.3 Plasmids

To construct Dnal1 and Dnal4 expression plasmids, cDNAs of Dnal1 and Dnal4 respectively were amplified by PCR (Platinum Hi-Fidelity TaqDNA polymerase, Invitrogen) from cDNA clones (BioSource). Dnal1 and Dnal4 sequences were further amplified using primers incorporating flag and myc epitope tagged sequences respectively and then subcloned into pCAG vector to create pCAG- Dnal1-flag and pCAG-Dnal4-myc plasmids. The GFP fused full length (pCAG- Dyx1c1-GFP) and deletion constructs of Dyx1c1 (pCAG-ΔTPR-GFP and pCAG- Δp23-GFP) were constructed as described previously124. The pCAG-GFP plasmid was used as a control. The flag-tagged DNAAF1, DNAAF2 and DNAAF3 plasmids were a gift from Dr. Heymut Omran. The sequence of all plasmids was confirmed by sequencing.

Human embryonic kidney (HEK293T) cells were grown to confluence in DMEM supplemented with 10% FBS, 1% non-essential amino acid (NEAA) and 1% penicillin-streptomycin antibiotics (Invitrogen). Cultures were plated at a density of 106 cells on a 10-cm petridish. DNA (24 µg) was added to the petridish with 75 µl of Lipofectamine 2000 reagent (Invitrogen) in OptiMEM-1. The transfection mix was added to cells in normal growth media and incubated for 48 h at 37 °C. To obtain the protein lysates from the cells, the normal growth media was removed and the cells were washed with PBS. The cells were then collected, centrifuged to pellet the cells and the pellet was incubated in RIPA buffer supplemented with 1X Proteinase Inhibitor Cocktail for 20 min on ice. The lysates were then centrifuged at 2000xg to pellet the debris. Protein blots were also performed on the transfected cells as described above.

5.5.5 Immunoprecipitation

Immunoprecipitation assays were performed using the Dynabeads Protein G Immunoprecipitation kit (Invitrogen). Briefly, Dynabeads were resuspended in the vial and separated on a magnet from solution. Antibody to Dyx1c1 (Sigma, SAB4200128), Dnal1 (Proteintech, 15809-1-AP), Dnal4 (Proteintech, 10388-1- AP), DNAI2 (Abnova, M01, clone 1C8) or GFP (Life Technologies, A11122) (5 µg) was diluted in 200 µl of Washing and Binding Solution and incubated with rotation for 120 min at room temperature. Bead-antibody complexes were separated on the magnet, washed by gentle pipetting and separated. Protein lysates were incubated with the bead-antibody complexes overnight at 4 °C. Bead-antibody-antigen complexes were then washed using the washing solution

three times. Complexes were then incubated with elution buffer for 10–15 min to dissociate them. Beads were separated on a magnet, and supernatant containing the proteins was separated by SDS-PAGE and analyzed by protein blotting using monoclonal antibody to DNAI2 (Abnova, M01, clone 1C8; 1:500 dilution), antibody to DNAI1 (Proteintech, 12756-1-AP; 1:500 dilution), antibody to Hsp70 (BD Biosciences, 610607; 1:1,000 dilution), antibody to Txndc3 (Proteintech, 13586-1-AP, 1:500), antibody to Hsp90 (BD Biosciences, 610418; 1:1,000 dilution), antibody to CCT4 (Aviva Systems Biology, ARP34271_P050; 1:500 dilution), antibody to CCT3 (Proteintech, 10571-1-AP; 1:500 dilution), anti- body to CCT5 (Proteintech, 11603-1-AP; 1:500 dilution), antibody to CCT8 (Proteintech, 12263-1-AP; 1:500 dilution), c-myc antibody (Sigma, C3956, 1:500 dilution), c-myc antibody mouse monoclonal (Sigma, M4439, 1:750 dilution), antibody to flag produced in rabbit (Sigma, SAB1306078, 1:1000 dilution), and mouse monoclonal anti-flag antibody (Sigma, F4042, 1:500 dilution). Rabbit IgG was used as a control.