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HEK293 cells were transfected with GPR56, Gα13 siRNA, scrambled siRNA, GPR56 ^C, GPR56^N, Cells at 30% confluency were serum starved for 24hrs and treated with testosterone for 24 hrs at 10nM and H89 for 30 mins at 30μM, GTP_ƳS was used as positive control. The cells were then lysed in 300μl of cell lysis buffer (provided with kit, 50 mM Tris p H 7.5, 10 mM MgCl₂ 0.5 M NaCl and 2% Igepal) per 90 mm dish. After centrifugation at 15,000 x g at 4°C for 10 min, 200 μl of supernatant was incubated with 30μg of rhotekin (GST-RBD) beads at 4°C for 1 hour on rocker. The resins are then washed twice with 500 μl of wash buffer (25 m M Tris p H 7.5, 30 mM MgCl₂, 40 mM NaCl and boiled for 2 min in Laemmli’s sample buffer and bands were detected using anti-RhoA mAb (cytoskeleton ARH03, 1:500) by femtoLUCENT (G Biosciences).

Whole cell lysate was prepared using cell lysis buffer containing 20mM Tris/HCl (pH 7.5), 150mM NaCl, 1% Nonidet P40, 1mM Dithiothreitol with protease inhibitors aprotinin (10μg), Phenyl methane Sulfonyl fluoride (10μg) and phosphatase inhibitor cocktail (10μl). The lysate was centrifuged at 14,000rpm at 4°C for 15 mins. The pellet was discarded, and supernatant was used as whole cell lysate. The protein concentrations were measured using Bradford kit (Himedia). The samples were separated by SDS/PAGE and transferred to PVDF membrane (MDI). The membrane was blocked in TBST (Tris buffered saline/Tween 20: 20mM Tris/HCl ph-7.5, 150mM NaCl, 0.1% Tween 20) with 5% Non-fat dry milk for 2 hours at 4°C. The membrane was incubated with primary antibody diluted in TBST with 3% non-fat dry milk overnight at 4°C. Membrane was washed thrice with TBST for 15 mins each and incubated with HRP-conjugated secondary antibody for 2 hours at RT. Post washing, immunoreactive bands were detected by femtoLUCENT (G Biosciences). Primary AR antibody (AR 441), Primary GPR56 antibody and HRP conjugate secondary antibody were purchased from Santa Cruz Biotechnology Ltd, Histone H3B antibody was a kind gift provided by Dr. Sandeep Saxena from National Institute of Immunology, India.