GST-pulldown

3.11.8.1 Purification of RhoH-GST fusion protein and GST

BL21(DE3) E. coli bacteria transformed with RhoH-GST or GST were inoculated in 2 ml LB (containing ampicillin and chloramphenicol) and grown o/n at 37°C with vigorous shaking. Next morning, 100 ml LB (containing ampicillin and chloramphenicol) were inoculated with 2 ml o/n culture and grown for 2 h at 37°C until OD600 reached 0.5-0.6. The bacteria were

cooled down for 15 min on ice and 100 µl 1 M IPTG (final concentration 1 mM) were added to bacteria to induce the protein production. The cells were grown for 6 h at 26°C or o/n at 18°C with shaking. For monitoring protein expression, small aliquot of bacteria suspensions were saved before IPTG induction and after protein production, centrifuged and resuspended in 4x sample buffer. After protein synthesis, the bacteria were centrifuged in 2x 50 ml tubes for 10 min at 4000 rpm, 4°C. The pellets were washed with 20 ml cold buffer A (50 mM Tris pH 7.4, 5 mM MgCl2, 50 mM NaCl), combined in one tube and centrifuged for 10 min at

4000 rpm, 4°C. The supernatant was removed and the pellet frozen on dry ice / EtOH bath and stored at -80°C. Alternatively, the pellet was directly processed without freezing. Bacteria were resuspended in 5 ml cold buffer A+ (buffer A, supplemented freshly with 1 mM PMSF, 1 mM DTT, protease inhibitor cocktail) (if the pellet was frozen, it was thawed at 37°C), sonicated 4x for 15 sec (Gerhard Heinemann, Laboratoriums-Ausrüstungen; small tip, output control 5) with 30 sec pausing and placing the tube on ice between the pulses. 50 µl Triton X- 100 (final concentration 1%) and the samples were tumbled for 20 min at 4°C. Then 550 µl glycerol (final concentration 10%) were added and the samples were distributed into 5-6 1.5 ml tubes. The lysates were cleared by centrifugation for 15 min at 14000 rpm, 4°C and the supernatants pooled. Bacterial extracts were aliquoted, frozen on dry ice / EtOH and stored at -80°C.

To bind GST proteins to glutathione sepharose (GS) beads Glutathion Sepharose 4 Fast Flow; Amersham Pharmacia Biotech; GS-beads), 40 µl of GS-beads slurry per sample were centrifuged for 1 min, 13000 rpm, 4°C, and washed 4x with 1 ml cold CLB+ buffer (50 mM Tris pH 7.4, 5 mM MgCl2, 200 mM NaCl, 1% NP40, 10% glycerol, freshly added 1 mM

PMSF, 2 mM Na3VO4, protease inhibitor cocktail) (centrifugation steps in between 1 min,

13000 rpm, 4°C) and incubated with 2 ml RhoH-GST bacterial extract for 1 h at 4°C on a rotary wheel. After binding, the GS-beads were washed 3x with 2 ml CLB+ buffer and kept on ice until incubating with the thymocyte extracts. In case with GST protein, 100 µl bacterial extract were used, and after binding the beads were washed 3x with 1 ml CLB+ buffer.

For monitoring the protein purification and binding to GS-beads, a small aliquot of bacteria lysate was resuspended in 4x sample buffer, the pellet after centrifugation, and the GS-beads after incubation with bacterial extracts were resuspended in 1x sample buffer, boiled at 95°C for 5 min and subjected to SDS-PAGE and Coomassie stain.

3.11.8.2 Pulldown

Since GEFs bind preferentially to the nucleotide-depleted state compared with GTP- or GDP- bound states, nucleotide-depleted condition was used to precipitate GEFs binding to RhoH. Nucleotide-loaded condition was used to precipitate effectors binding to RhoH.

Wild type thymocytes were stimulated as described in 3.11.6.1 or left non-stimulated and 6x107 cells were lysed in 600 µl lysis buffer: 50 mM Tris pH 7.4, 100 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM Na3VO4, 100 mM NaF, containing a protease inhibitor cocktail

supplemented either with 2 mM EDTA (nucleotide-depleted condition) to chelate magnesium, an essential co-factor for nucleotide binding, or 5 mM MgCl2 (nucleotide-loaded condition) to

maintain nucleotide binding. The thymocyte lysates were pre-cleared with 100 µl GS-beads for 1 h at 4°C on a rotary wheel to remove extract components which bind non-specifically to GS-beads. The GS-beads were washed before incubation with thymocyte lysate 3x with 1 ml lysis buffer.

Meanwhile, recombinantly expressed GST and RhoH-GST attached to GS-beads (s. 3.11.8.1) were incubated with 1 ml 50 mM Tris pH 7.5, 1 mM DTT, supplemented either with 5 mM EDTA or 5 mM MgCl2 and 40 µM GTPγS (Sigma) for 10 min at 30°C. After centrifugation

step for 1 min at 13000 rpm, 4°C, the beads were incubated with corresponding thymocyte lysate for 4 h or o/n at 4°C on a rotary wheel.

The GS-beads were washed 3x with the lysis buffer (supplemented either with EDTA or MgCl2), resuspended in 40 µl 1x sample buffer and denatured at 95°C for 5 min.

3.11.9 Myc-IP

Non-transfected 293 cells and 293 cells, transfected with RhoH-myc via calcium phosphate method (s. 3.7.6.1), were harvested 48 h later: the cells were trypsinized (s. 3.7.3) and lysed in 400 µl lysis buffer 50 mM Tris pH 7.4, 100 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM Na3VO4, 100 mM NaF, containing a protease inhibitor cocktail, supplemented either

with 2 mM EDTA (nucleotide-depleted condition) or 5 mM MgCl2 (nucleotide-loaded condi-

tion). A small aliquot was saved for later analysis of transfection efficiency in Western blotting.

Meanwhile, IgG beads (40 µl slurry per sample) were washed 3x with 1 ml lysis buffer, supplemented either with 2 mM EDTA or loaded with 5 mM MgCl2 and 40 µM GTPγS (s.

3.11.8.2) and incubated with the protein extract of 293 cells and 4 µg 9E10 antibody (Upstate), cortactin (4F11, Upstate) antibody or no antibody for 2 h at 4°C on a rotary wheel. After binding, the IgG beads were washed 3x with 1 ml lysis buffer, containing either EDTA or MgCl2, and rocked with lysate of non-stimulated or stimulated thymocytes (s. 3.11.6.1) o/n

at 4°C. Subsequent steps including washing and resuspending the IgG beads in 1x sample buffer were carried out according to 3.11.8.2.

3.11.10 Flag-IP

1x107 Jurkat TAg cells were harvested 48 h after DMRIE-C transfection (s. 3.7.6.2) of RhoH- Flag or empty PS080 vector and stimulated with F101.01 as described in 3.11.6.2. After stimulation the cells were washed with 500 µl cold PBS, centrifuged for 5 min, 300xrcf, 4°C and lysed in 300 µl 50 mM Tris pH 7.4, 100 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM Na3VO4, 100 mM NaF, containing a protease inhibitor cocktail, supplemented either

with 2 mM EDTA or 5 mM MgCl2 for 20 min on ice. The lysates were cleared by centrifu-

gation at 14000 rpm for 15 min at 4°C. 30 µl were denatured with 4x sample buffer at 95°C and stored at -20°C for later analysis of stimulation and transfection status. Meanwhile, 40 µl anti-Flag M2 beads (Sigma) were washed with 3x with 1 ml cold TBS. The centrifugation steps were carried out for 1 min, 10000 rpm, 4°C. The protein extract was added to the beads and rotated for 2 h at 4°C. After binding of RhoH-Flag to the beads, the samples were centrifuged for 1 min, 10000 rpm, 4°C, the flow through was saved for later analysis of binding efficiency of RhoH-Flag to the beads and the beads were washed 3x with 1 ml cold TBS. Next, the beads were either denatured in 40 µl 1x sample buffer at 95°C or the RhoH- Flag and putative associated proteins were eluted from anti-Flag M2 beads by competing with 6 µl 5 µg/ml 3xFlag peptide (Sigma) in 50 µl TBS for 30 min on ice with gentle mixing every 2 min. Then the beads were centrifuged for 1 min, 10000 rpm, 4°C and the eluat was denatured in 4x sample buffer at 95°C. The elution efficiency was controlled by analyzing the beads, denatured in 1x sample buffer at 95°C, on SDS-PAGE.

3.11.11 TAP-tag-pulldown

1x107 _{Jurkat TAg cells were harvested 48 h after DMRIE-C transfection (s. 3.7.6.2) of RhoH-}

TAP-tag, RhoH-N62L-TAP-tag, RhoH-T18N-TAP-tag or empty pCMV-N-terminal-TAP-tag vector and stimulated with F101.01 as described in 3.11.6.2. After stimulation the cells were washed with 500 µl cold PBS, centrifuged for 5 min, 300xrcf, 4°C and lysed in the same lysis

buffer, supplemented either with EDTA (nucleotide-depleted condition) or MgCl2 (nucleo-

tide-loaded condition), which was used for Flag-IP (s. 3.11.10). 30 µl were denatured with 4x sample buffer at 95°C and stored at -20°C for later analysis of stimulation and transfection status.

Meanwhile, 40 µl of IgG sepharose 6 Fast Flow beads (GE Healthcare/Amersham Pharmacia Biotech) were washed with 1 ml HAc (adjusted to pH 3.4 with NH4Ac), centrifuged for 1

min, 6000 rpm, 4°C, washed with 1 ml TBS, washed again with 1 ml Hac pH 3.4 and 1 ml TBS. The beads were subsequently equilibrated with 3x 1 ml lysis buffer, containing either EDTA or MgCl2, and blocked with 1 ml 1% BSA in lysis buffer at 4°C on a rotary wheel.

After blocking, the beads were washed 3x with 1 ml lysis buffer, containing either EDTA or MgCl2. The Jurkat TAg protein extract was added to the beads and the samples were rotated

for 2 h at 4°C. Next, the samples were centrifuged for 1 min, 6000 rpm, 4°C, the flow through was saved for later analysis of binding efficiency of RhoH-TAP-tag to the beads and the beads were washed 3x with 1 ml cold lysis buffer, supplemented with EDTA or MgCl2. The

beads were resuspended in 40 µl 1x sample buffer and denatured at 95°C for 5 min.

3.12 Microscopy

Cell and tissue morphology was analysed with phase contrast microscope Axioskop + Leica DC500 (Zeiss). Fluorescence microscopy was done with Leica DMRA2 + Hamamatsu ORCA-ER camera (Leica) and confocal Leica DMIRE2 + Leica TSC SP2 (Leica).

3.13 Statistical evaluation

All averages are shown with standard deviation. Student's t-test (p) was performed to assess the significance of observed differences. Differences with p<0.05 are significant.

4 Results