Co-immunoprecipitation (Co-IP), and western blot

Cell extracts from O9-1 cells transfected with control or Smarcc1 (BAF155)/Smarcc2 (BAF170) mix siRNA pool were prepared using cold RIPA buffer (Thermo Fisher Scientific, catalog no. 89901) supplemented with Halt protease and phosphatase inhibitor cocktail (100X) (Thermo Fisher Scientific, catalog no. 78440). Protein concentration was checked with spectrophotometry as compared to standard BSA solutions. For the co-immunoprecipitation experiment, cell extracts were then incubated with control IgG, anti-Tead antibody (1:100 dilution; Cell Signaling, Catalog no. 13295S) or anti-Brg1 antibody (1:100 dilution, Abcam, Catalog no. ab-70558) at 4°C overnight. The next day, 20–30μL of protein A-agarose beads (Santa Cruz, Catalog no. sc-2001) were added into the cells extracts and further incubated at 4°C for 2 hours on a roller. After a quick spin, the supernatant was removed and agarose beads were washed thrice with 1mL of cold PBS supplemented with inhibitors. Finally, immunoprecipitated proteins were extracted in Laemmli sample buffer (BIO-RAD, Catalog no.1610737) and evaluated with western blot analysis. Input western blots were also performed with 10% of the total protein used for the Co-IP experiment. Western blotting experiments were performed as described previously [68–71]. The primary antibodies used include anti-BAF155 (1:300 dilution, Santa Cruz, Catalog no. sc-32763), anti-BAF170 (1:300 dilution, Santa Cruz, Catalog no. sc-17838), anti-β-actin (1:1000 dilution, Santa Cruz, Catalog no. sc-47778), anti-Brg1 (1:1000 dilution, Abcam, Catalog no. ab-70558), anti-Tead (1:1000 dilution, Cell Signaling, Catalog no. 13295S) and anti-Yap (1:300 dilution, Santa Cruz, Catalog no. sc-376830). The secondary antibodies used include rabbit anti-mouse IgG H&L (HRP) (1:5000 dilution, Abcam, Catalog no. ab97046) and goat anti-rabbit IgG H&L (HRP) (1:5000 dilution, Abcam, Catalog no. ab6721).