Bp1600

Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences 15710) for 20 min and permeabilized in 0.01% Triton X100 for 1 h at room temperature. Cells were blocked in 5% BSA (Sigma BP1600) for 1 h, and primary antibodies added and incubated at 4°C overnight. Primary antibodies used are as follows in 5% BSA: aSMA (abcam ab7817 1:1000), YAP (Santa Cruz Biotechnology sc‐101199, 1:500), Methylated Lysine (Novus Biologics NB600‐824, 1:500), Acetylated Lysine (abcam ab190479, 1:300), Histone H3 acetylation (EMD Millapore 06‐599, 1:1000), and CREBBP (abcam ab2832, 1:1000). Cells were washed, and secondary antibodies were added for 30 min. Cells were imaged with automated Perkin Elmer Operetta. Cell area, nuclear area, antibody intensity was quantified using Perkin Elmer's Harmony software. For αSMA measurements, values were normalized to Cellmask intensity.

Cells were seeded to 6cm plates at 5x10⁵ cells per plate and allowed to incubate overnight. The following day, plates were washed once with ice cold 1xPBS, then lysed with a rubber policeman and ice cold RIPA buffer (150mM NaCl, 5mM EDTA, 50mM Tris pH 8.0, 1% NP-40) with 1mM phosphatase (ThermoScientific, 88667) and 1mM protease (Roche, 04693159001) inhibitor cocktails added. The following antibodies were used: FLAG (Sigma, F3165) at 1:50[17 (link)]00 dilution in 5% BSA (Fisher, BP1600) in TBST; GAPDH (Sigma, G8795) at 1:5000 dilution in 5% BSA in TBST.