Rabbit anti phospho stat1 tyr701 ab

Lung protein was extracted as described above. Proteins were separated on stain-free gels (Bio-rad) and transferred onto polyvinylidene fluoride (PVDF) membranes and incubated with rabbit anti-STAT1 Ab (1:2000 dilution, Cell Signaling Technology) or with rabbit anti-phospho-STAT1 (Tyr701) Ab (1:2000 dilution, Cell Signaling Technology). Secondary Abs were goat anti-rabbit IgG (1:5000 dilution, Abcam). Anti-β-actin (1:2000 dilution, Biolegend) was incubated after stripping the STAT1 or P-STAT1 Ab. The relative amount of STAT1 and P-STAT1 protein was quantified by normalizing the amount of β-actin housekeeping protein using Image Lab software 6.1.

Cells were lysed on ice with RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) containing protease inhibitor cocktail (Sigma-Aldrich). Protein content of the lysates was determined using the Bradford Protein Assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts (40 μg/lane) of protein were separated by 12 % SDS PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The blots were incubated with rabbit anti-PCNA antibody (Ab), rabbit anti-phospho-STAT1 (Tyr701) Ab (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-S100A4 Ab (Abcam, Cambridge, UK), mouse anti-β-actin Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-fibronectin Ab mouse anti-β-catenin Ab (Santa Cruz Biotechnology), mouse anti-STAT1 Ab, and mouse anti-c-fos Ab (Abcam). Primary Ab binding was detected with secondary antibody (anti-mouse or anti-rabbit HRP-conjugated IgG secondary antibody) that was detected using enhanced chemiluminescence substrate kit (GE Healthcare, Piscataway, NJ, USA). Quantification was performed with a ChemiDoc TM XRS + scanner and Image Lab Software (Bio Rad, CA, USA). The densities of each sample were normalized to the β-actin.