Anti phosphorylation p38

Mouse adipose tissue was homogenized with a tissue homogenizer at centrifuge of 12000 g at 4 °C for 10 min using RIPA lysis buffer containing protease inhibitor (Beyotime Biotechnology) and phosphatase inhibitor (MedChemExpress).The supernatant was aspirated, and the protein concentration was measured by the BCA Protein Assay Kit (Vazyme Biotech Co., Ltd.).The protein sample was loaded for polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane in an electrophoresis transfer buffer containing 10% methanol. The membrane was blocked with 5% BSA (Sigma-Aldrich) for 2 h at room temperature, followed by incubation overnight at 4 °C with anti-p38(1:500; Proteintech™), anti-phosphorylation p38(1:1000;Beyotime), anti-ERK1/2(1:2000; Proteintech), anti-phosphorylation ERK1/2(1:1000; Beyotime), anti-JNK(1:3000; Proteintech™), anti-phosphorylation JNK (1:1000; Beyotime), and anti-GAPDH (1:50,000; Proteintech Wuhan, China) antibodies. After overnight incubation, the blots were incubated with the corresponding secondary antibodies (1:10,000; Bioworld) for 1 h at room temperature. Bands of target proteins were visualized using an enhanced chemiluminescence kit (Vazyme Biotech Co.,Ltd).

Proteins from cells were lysed with a RIPA lysis buffer containing 1 mM PMSF (Beyotime) referring to the manufacturer’s instruction. Then, the concentration was measured by BCA Protein Assay Kit (Beyotime). The proteins were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Sigma-Aldrich). The membranes were blocked in 6% skim milk for 2 h and then incubated with primary antibodies at 4 °C overnight and finally with the secondary antibody (1:6000; Bioworld, Nanjing, China) for 1 h. The following primary antibodies were used: anti-GAPDH (1:6000; Proteintech™, WuHan, China), anti-RUNX2 (1:1000; Cell Signaling Technology, MA, USA), anti-OPN (1:1000, Proteintech™), anti-ALP (1:2000; Proteintech™), anti-COLLAGEN type I (1:2000;Proteintech™), anti-SMAD2(1:1000; Cell Signaling Technology), anti-phosphorylation SMAD2 (1:1000; Beyotime), anti-SMAD3 (1:1000; Beyotime), anti-phosphorylation SMAD3(1:1000; Beyotime),anti-ERK1/2(1:2000; Proteintech™), anti-phosphorylation ERK1/2(1:1000; Beyotime), anti-JNK (1:3000; Proteintech™), anti-phosphorylation JNK (1:1000; Beyotime), anti-P38(1:500; Proteintech™), and anti-phosphorylation P38(1:1000; Beyotime). The visualization of the protein bands was used with enhanced chemiluminescence kit (Beyotime).