Phospho stat3

HRECs were lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Sigma Aldrich, St. Louis, MO, USA). The cell lysates were subjected to Western blot analysis as described previously (Sharma et al., 2007b (link); Sharma et al., 2010 (link); Sharma et al., 2008 (link)). Samples containing 20–40 µg protein were separated on a 4–20% SDS polyacrylamide gel. The proteins were then transferred to a nitrocellulose membrane using the Trans-Blot® Turbo^™ Transfer System (BioRad; Hercules, CA, USA). The membrane was then blocked in 5% bovine serum albumin (BSA) and incubated in primary antibody overnight at 4°C. Blots were then washed in TBST and incubated with the appropriate secondary antibody labeled with HRP. The bands were visualized with chemiluminescent substrate (Pierce Rockford, IL) and exposed on CL-Xposure^™ X-ray film (Pierce Rockf ord, IL). Antibodies used were: phospho-STAT3 (1:500, Millipore Darmstadt, Germany), STAT3 (1:2000, Millipore Darmstadt, Germany), gp130 (1:200, Cell Signaling Danvers, MA, USA), IL6R (1:500, Santa Cruz Biotechnology, Dallas, Texas), ICAM-1 (1:500, Cell Signaling Danvers, MA, USA) and β-actin (1:5000, Cell Signaling Danvers, MA, USA). β-actin was used as loading control.