About 5 × 106 of transfected cells were lysed for 30 min in 1 ml of IP buffer containing 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP-40, 0.25% sodium deoxycholate, 2 mM EDTA, and 1 mM Na3VO4, supplemented with protease inhibitors. For IPs, lysates were cleared by centrifugation (14,000g for 30 min), and supernatants were incubated for 1 hour with antibodies on rotating platform at 4°C. Immunocomplexes were collected on 30 μl of A/G agarose (Santa Cruz Biotechnology) in overnight incubation, spun down, and washed four times in 400 μl of the IP buffer. Proteins attached to the beads were eluted to 70 μl of loading buffer. For Western blotting, 20 μl of cell lysates or immunoprecipitates were resolved by SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, blocked with 5% (w/v) in tris-buffered saline (TBS)/Tween 20] nonfat milk, incubated with primary antibodies overnight at 4°C, washed 3× for 10 min in TBS/Tween 20, incubated with secondary antibodies at room temperature for 1 hour, washed 3× for 10 min TBS/Tween 20, and visualized by chemiluminiscence using Pierce ECL (Thermo Fisher Scientific), Immobilon Western (Millipore), or SuperSignal West Femto (Thermo Fisher Scientific) substrates. Table S2 lists all antibodies used in the study.

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