Anti phospho stat3 tyr705

Proteins were extracted from mouse kidney cortex and cultured cells with RIPA lysis buffer (Beyotime, Jiangsu, China). Protein concentration was measured by BCA Protein Assay kit (Bio‐Rad, Hercules, CA, USA). After boiling for 5 min. at 95°C in 5× loading buffer, an equal amount of protein (40 μg) was separated via 8% SDS‐PAGE and electrotransferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). The membranes were routinely processed via blocking with 5% milk or 5% BSA and were incubated overnight with primary antibodies, followed by a 1‐hr incubation with horseradish peroxidase‐conjugated secondary antibodies. Immunoreactive proteins were detected using an Enhanced Chemiluminescence (ECL) Kit (Amersham). The antibodies used in this study were as follows: anti‐mIL‐6R (rabbit, 1:200), anti‐gp130 (rabbit, 1:1000), anti‐phospho‐STAT3 Tyr705 (mouse, 1:200) and β‐actin (mouse, 1:10,000) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐sIL‐6R (mouse, 1:500; Abcam, Cambridge, MA, UK); and anti‐desmin (rabbit, 1:1000), anti‐phospho‐STAT3 Ser727 (rabbit, 1:1000) and anti‐STAT3 (rabbit, 1:1000) from Bioworld Technology (Louis Park, MN, USA). Densities of blots were measured by ImageJ software (NIH, Bethesda, MD, USA).

The following primary antibodies were used for Western blot analysis: rabbit polyclonal anti-PARP1(1:1000) (Proteintech, Rosemont, IL, USA, #13371-1), rabbit polyclonal anti-phospho STAT3 Tyr705 (1:500) (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, #sc-8059), mouse monoclonal anti-STAT3 (1:100) (BD Transduction Lab, Franklin Lakes, NJ, USA, #610189), rabbit polyclonal anti-LC3I/II (1:1000) (Novus, CO, USA, #NB100-2220), mouse monoclonal anti-p62/SQSTM1 (1:300) (BD Transduction Lab, #610832), rabbit polyclonal anti-XBP1 (1:1000) (NovusBio, #NBP1-77681SS), rabbit polyclonal anti-ATF6 (1:200) (Cell Signaling Technology, Danvers, MA, USA, #65880), rabbit polyclonal anti-phospho eIF2α (Ser15) (1:200) (Cell Signaling, #3398), rabbit polyclonal anti-eIF2α (1:500) (Cell Signaling, #9722), mouse monoclonal anti-β-actin (1:10000) (Sigma Aldrich, #A5441) and anti hsp70 (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, #sc-32239) were used as loading control. Goat anti-rabbit IgG-horseradish peroxidase HRP (1:10000) (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, sc-2004), goat anti-mouse IgG-horseradish peroxidase HRP (1:10000) (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, sc-2005) were used as secondary antibodies. All primary and secondary antibodies used in this study were diluted in a PBS-0.2% Tween 20 solution containing 3% BSA.