Nuclear cytoplasm extraction kit

The following primary antibodies were used for immunoblot analysis: YAP, phospho-YAP (Ser127), Src, Lamine-b and PD-L1 from Cell Signaling, Inc. (Danvers, MA); phospho-YAP (Tyr357) from Abcam (Cambridge, MA) and Sigma-Aldrich (St. Louis, MO); TAZ and α-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA). Total protein was extracted from cell lines using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Rockford, IL), and nuclear/cytoplasm proteins extracted using a nuclear/cytoplasm extraction kit (Thermo Fisher Scientific Inc.) were supplied with Complete Protease Inhibitor Cocktails (Roche, Lewes, UK), according to the manufacturers’ protocols. The protein concentrations were measured with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL). A total of 15 μg of proteins was run on 4∼20% gradient SDS–polyacrylamide gels (Bio-Rad) and transferred to Immobilon-P nitrocellulose membranes (Millipore, Bellerica, MA). The membranes were blocked in 5% non-fat milk and then probed with the primary antibodies overnight at 4°C. The membranes were incubated with appropriate secondary antibodies, and detected by using an ECL blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ).

The primary antibodies for CDK7, YAP, phosphor‐YAP (Ser127), cyr61, CTGF and Tead4 used in Western blot analysis were purchased from Cell Signaling, Inc. NF/Merlin antibody was purchased from LSBio. Total protein was extracted from cell lines using the Mammalian protein extraction kit (M‐PER, Thermo), and nuclear/cytoplasm proteins were extracted using a nuclear/cytoplasm extraction kit (Thermo Fisher Scientific Inc) according to the manufacturers' protocols. The protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc). Total protein for each sample was 15 μg, and the samples were run on 4%‐20% gradient SDS‐polyacrylamide gels (Bio‐Rad Laboratories, Inc). After proteins were transferred to Immobilon‐P nitrocellulose membranes (Millipore), the membranes were blocked in 5% non‐fat milk and then probed with the primary antibodies overnight at 4°C. The membranes were incubated with HRP‐conjugated secondary antibodies for 1 hour at room temperature and detected by using an ECL blotting analysis system (Amersham Pharmacia Biotech). We performed the experiments three times.