Western lighting plus ecl detection reagent

Protein (50 µg) was resolved on 8% Express-Plus PAGE gels in Tris-MOPS (SDS) running buffer (GenScript, Piscataway, NJ, USA), transferred to PVDF membranes and incubated at 4°C overnight with primary antibodies: CDK8 (sc-1521, SantaCruz, Santa Cruz, CA, USA), CDK19 (HPA007053, Sigma-Aldrich), ER (sc-543, SantaCruz), phospho-ER Ser118 (sc-101675, SantaCruz) and GAPDH (#5174, Cell Signaling Technology, Danvers, MA, USA) followed by either anti-goat (sc-2020, SantaCruz), anti-rabbit (#31460, ThermoFisher Scientific) or anti-mouse (31430, ThermoFisher Scientific) secondary antibodies. Bands were visualized with Western Lighting Plus ECL detection reagent (Perkin Elmer, Waltham, MA, USA) using ChemiDoc Touch™ (BioRad). Images were analyzed and densitometry performed using ImageLab software (Biorad).

Cells grown and treated in P100 plates were washed with cold PBS twice and then lysed in RIPA lysis buffer (50 mM Tris–HCl, pH 8.0; 150 mM NaCl; 5 mM EDTA; 0.5 mM EGTA; 1% Igepal CA-630 (NP-40); 0.1% SDS; 0.5% Na deoxycholate) or IP lysis buffer (10 mM Tris–HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1% Igepal CA-630) supplemented with 1x protease/phosphatase inhibitor cocktail (Thermo-Fisher Scientific #78438), 2 mM Na₃VO₄ and 10 mM NaF. Ice-cold lysate was briefly sonicated to solubilize chromatin proteins before centrifugation. Protein concentrations were determined using the DC protein assay (Bio-Rad). Lysate samples with the same amount of total protein (40–50 μg) were mixed with 4× Laemmli Sample Buffer (Bio-Rad, with 2-mercaptoethanol) and run on 4–12% Express-Plus PAGE gels in Tris–MOPS (SDS) running buffer (GenScript). Proteins were transferred to PVDF membranes, blocked with 5% non-fat milk and incubated with primary and then secondary antibodies (detailed information is in Supplementary Table S1). Bands were visualized with Western Lighting Plus ECL detection reagent (Perkin Elmer, Waltham, MA, USA) using ChemiDoc Touch™ (Bio-Rad). Image processing and densitometry analysis were performed using ImageLab software (Bio-Rad).

Cells were cultured in 60-mm dishes and treated under different conditions before being lysed in RIPA lysis buffer with protease/phosphatase inhibitor cocktail. The protein concentration of extracts was determined using the DC protein assay (Bio-Rad Laboratories). Protein samples (50 μg) were resolved on 4–12% Express-Plus PAGE gels in Tris-MOPS (SDS) running buffer (GenScript, Piscataway, NJ, USA), transferred to PVDF membranes and incubated at 4 °C overnight with primary antibodies STAT1 (sc-592, Santa Cruz Biotechnology, Dallas, TX, USA) and pSTAT1-Ser727 (#8826, Cell Signaling Technology, Danvers, MA, USA) followed by anti-rabbit (NA934, GE Healthcare, Chicago, IL, USA) secondary antibodies. Bands were visualized with Western Lighting Plus ECL detection reagent (Perkin Elmer, Waltham, MA, USA) using ChemiDoc Touch™ (Bio-Rad). Images were analyzed using ImageLab (Bio-Rad) and ImageJ (version 1.52p) software for protein signal quantification.