Anti bag3

Cells were lysed with NP40 Buffer (ThermoFisher) supplemented with Halt protease and phosphatase inhibitors (ThermoFisher) for 10 min on ice. Protein extracts were quantified with a micro BCA kit (BioRad) and cell lysate was denatured in Laemmli SDS Buffer (1x, Alfa Aesar) at 95 °C for 5 min. 5–10 μg of protein was loaded into precast gels (4–12% mini-PROTEAN TGX, BioRad), ran for ~1 h at 120V, and then transferred to nitrocellulose blotting membranes (0.45um, GE Healthcare) for 1.5 h at 0.4 A at 4 °C using standard protocols. Blotting membranes were blocked with skim milk powder (5%) in tris buffered saline with Tween® 20 (TBST, 0.05% w/v Tween® 20) for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C in 5% BSA in TBST. The membranes were then washed 3x with TBST for 10 min. Secondary antibodies were diluted in 5% BSA in TBST and incubated for 1 h at room temperature. The following primary and secondary antibodies were used: anti-BAG3 (1:5000, ProteinTech), anti-Histone H3 (1:10000, Abcam), anti-YAP (1:2000, Cell Signaling), anti-pYAP (1:2000, Cell Signaling), antimouse-HRP (1:5000, Jackson ImmunoResearch), and antirabbit-HRP (1:5000, Jackson ImmunoResearch). The ECL substrate (Pierce West Dura, ThermoFisher) was added and the chemiluminescence was captured using an ImageQuant LAS 4000 detector.