Anti phospho stat1 tyr 701 anti phospho stat3 tyr 705

Protein levels of MMP-1 and MMP-3 were assessed by Western blot analysis, as we described previously [40 (link)]. Briefly, cell lysates, prepared in radioimmunoprecipitation assay buffer, were resolved by electrophoresis on 4–12% Bis-Tris protein gel (Invitrogen, Carlsbad, CA, USA). Resultant bands were blotted onto polyvinylidene difluoride membranes, probed with anti-MMP-1 and MMP-3 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-STAT1 (Tyr-701) anti-phospho-STAT3 (Tyr-705) (Cell Signaling Technology, Boston, MA, USA), and anti-human β-actin (Sigma-Aldrich), and detected using enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA). The intensity of bands was measured with a LAS-3000 (Fujifilm, Tokyo, Japan).

The analysis of protein expression was performed as described previously [22 (link)] on cell lysates obtained with LDS sample buffer (Thermo Fisher Scientific). An amount of 20 µg of proteins were separated on Bolt™ 4–12% Bis-Tris mini protein gel (Thermo Fisher Scientific) and transferred to PVDF membranes (Immobilon-P membrane, Merck). Membranes were incubated for 1 h at RT in blocking buffer (Tris-buffered saline solution—TBS; 50 mM Tris-HCl pH 7.5, 150 mM NaCl) added with 3% non-fat dried milk and then overnight at 4 °C with primary antibodies in TBST (TBS + 0.5% Tween) containing 5% BSA. Anti-phospho-NF-κB p65 (Ser536), anti-phospho-IκBα (Ser32/36) anti-phospho-STAT1 (Tyr701), anti-phospho-STAT3 (Tyr705) and anti-phospho-c-Fos (Ser32) (1:2000, Cell Signaling Technology) rabbit polyclonal antibodies were employed. Anti-vinculin mouse monoclonal antibody (1:2000, Merck) was used as loading control. Immunoreactivity was visualized with SuperSignal™ West Pico PLUS Chemiluminescent HRP Substrate (Thermo Fisher Scientific). Western Blot images were captured with iBright FL1500 Imaging System (Thermo Fisher Scientific) and analyzed with iBright Analysis Software (Thermo Fisher Scientific).