Cell lysates, immunoprecipitates, and protein pulldowns were loaded and separated on SDS-polyacrylamide gel electrophoresis, transferred to Immobilon-P membranes (Millipore, catalog no. IPV00010), blocked with 5% milk/1× tris-buffered saline (TBS)–0.05% Tween, and incubated overnight at 4°C on a shaker, with primary antibodies directed against gp100 (Abcam, ab137078), β-actin (Santa Cruz Biotechnology, sc-47778), 6× His-tag (Sigma, H-1029), HA-tag (Covance), Gαq (Santa Cruz Biotechnology, sc-392), pYAP Ser127 (Cell Signaling Technology, 4911), YAP (Cell Signaling Technology, 4912), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, D16H11). After washing three times with TBS–0.05% Tween, membranes were incubated for 1 hour at room temperature with secondary antibodies (anti-rabbit, ABIN102010, dilution 1:10,000, antibodies-online GmbH, Aachen, Germany; or anti-mouse, dilution 1:5000, KPL, catalog no. 074-1802). Last, filters were washed with TBS-Tween and revealed using Amersham ECL Prime Western Blotting Detection Reagent Kit (GE Healthcare, Chicago, USA, product no. RPN2232) as per the manufacturer’s instructions.

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