Western Blot Analysis of STAT1 and Phospho-STAT1 in RA-FLS

The cell lysate was prepared from RA control and E2-treated RA-FLS through lysis in Radio-Immunoprecipitation Assay (RIPA) buffer (SIGMA) for Western blotting. Protein concentration was measured using BCA assay (G-biosciences) [45 (link)]. The 20 µg protein was resolved on 10% SDS-PAGE gel and then transferred to a nitrocellulose (NC) membrane at 20 V for 50 min using a semi-dry transfer unit (Bio-Rad, Hercules, CA, USA) followed by overnight blocking at 4 °C with 5% bovine serum albumin (BSA) in 1X Tris-buffered saline-tween 20 (TBST) [44 (link)]. The membrane was then incubated with primary antibodies STAT1 (9172T, Cell Signaling), Phospho-STAT1 (Tyr701) (9167L, Cell Signaling) (dilution 1:5000) overnight at 4 °C followed by washing and incubation with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (dilution 1:8000) (Jackson, West Grove, PA, USA) for 1 h at room temperature. The band intensity was measured by chemiluminescence (ECL) reagent (Thermo Scientific, Rockford, IL, USA). The band intensity was normalized by the housekeeping protein (β-actin; sc-47778) as a loading control.