Optimized Whole Cell and Nuclear Protein Extraction

Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described (Chen et al., 2020a (link),b (link),c (link); Wu X. et al., 2020 (link); Yang et al., 2020b (link); Zhang et al., 2020 (link); Chen B. et al., 2021 (link); Dong et al., 2021 (link)). Nuclear proteins were extracted using the NE-PER Kit (Pierce) following manufacturer’s recommendation. Specific antibodies or pre-immune IgGs were added to and incubated with cell lysates overnight before being absorbed by Protein A/G-plus Agarose beads (Santa Cruz). Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Western blot analyses were performed with anti-Clca2 (Proteintech, 19273-1, 1:500), anti-α-SMA (Sigma, A2547, 1:8000), anti-collagen type I (Proteintech, 14695-1, 1:2000), anti-Twist1 (Proteintech, 25465-1, 1:500), anti-HDAC1 (Santa Cruz, sc-7872, 1:1000), anti-HDAC2 (Santa Cruz, sc-7899, 1:1000), anti-HDAC3 (Santa Cruz, sc-11417, 1:1000), anti-HDAC8 (Santa Cruz, sc-11405, 1:1000), anti-FLAG (Sigma, F1804, 1:5000), and anti-β-actin (Sigma, A2228, 1:4000) antibodies.