Anti p38

Total protein was obtained using a Protein Extraction Kit (KeyGen BioTECH, Nanjing, China). SDS–PAGE separates proteins of different sizes and electroporates them onto PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% skimmed milk and incubated with anti-ZO-1(ER41204), anti-Occludin(R1510-33), anti-Claudin 1(ER1906-37), anti-Cleaved caspase 3(ET1602-47), anti-Bax(ER0907), anti-Bcl-2(ER1802-97), anti-iNOS(ER1706-89), anti-Arg1(ET1605-8), anti-p-p38(ER2001-52), anti-p38(ET1602-26), anti-p-JNK(ET1609-42), anti-JNK(ET1601-28), anti-p-ERK1/2(ET1603-22), anti-ERK1/2(ET1601-29), and anti-β-actin(R1207-1) antibodies at 4°C overnight(HUABIO, Hangzhou, China). After being washed with Tris Buffered Saline with Tween20 (TBST), the membranes were incubated with secondary antibodies for 1 h. Protein bands were visualized with an enhanced chemiluminescence (ECL) assay kit (Biosharp, Hangzhou, China) and measured with ImageJ software (NIH, Bethesda, MD, USA).

The protein levels of RUNX2, BSP, OPN, BAMBI, Smad5, p38, p-Samd5, and p-p38 were detected by Western blot. Total cell proteins were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma). BCA Protein Assay Kit (Beyotime) was used to determine the protein concentration. An equal volume of samples was placed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes (Millipore). Primary antibodies against anti-RUNX2 (1:1000; Abcam), anti-OPN (1:1000; Huabio), anti-BSP (1:1000; Abcam), anti-Smad5 (1:1000; Huabio), anti-p-Smad5 (1:1000; Huabio), anti-p38 (1:1000; Huabio), anti-p-p38 (1:1000; Huabio), and GAPDH (1:1000; Abcam) were incubated with the PVDF membranes at 4°C overnight. The membranes were incubated with secondary antibodies (1:5000; Abcam) for 2 h after washed with TBST. All the protein bands were normalized to GAPDH band.