Anti phospho stat3 ser727

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).

RAW264.7 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). WKYMVm and WRWWWW (WRW4) with purity > 95% was synthesized by GL Biochem and was dissolved in acetonitrile to prepare stock concentrations of 0.1, 1, 2, 5 and 10 μmol/L. Dulbecco's Modified Eagle's Medium (DMEM), alpha minimum essential medium (α‐MEM) and foetal bovine serum (FBS) were purchased from Invitrogen‐Gibco. RANKL and soluble mouse recombinant M‐CSF were obtained from R&D Systems. The Acid Phosphatase, Leukocyte (TRAP) assay kit and Actin Cytoskeleton and Focal Adhesion (FAK) Staining Kit were purchased from Sigma (Sigma‐Aldrich) and Merck, respectively. The Cell Counting Kit‐8 (CCK‐8) was purchased from Beyotime Biotechnology. Anti‐phospho‐NF‐kBp65, anti‐NF‐kBp65, anti‐STAT3, anti‐phospho‐STAT3 (Ser727), anti‐CTSK, anti‐c‐Fos, anti‐NFATc1, anti‐CD9, anti‐gp130, anti‐IL6 and anti‐ß‐actin antibodies were purchased from Bioworld Technology. Mouse IL‐1β, IL‐6 and TNF‐α ELISA kits were procured from Novus.