Gtx 100665

Cells were collected by centrifugation, washed in PBS, and lysed in RIPA buffer containing protease and phosphatase inhibitors (Thermo 78442) with vortex vigorously. Lysates were then centrifuged at 11,000g at 4 °C for 10 min and the supernatant (quantify the concentration first) was transferred into 4× diluted Laemmli sample buffer containing 2-mercaptoethanol and boiled in 100 °C for 5 min. After that, lysates were separated on 10% Bis-Tris polyacrylamide gels and transferred to PVDF membrane (Millipore IPVH85R). The blots were incubated with primary antibodies overnight at 4 °C and then with secondary antibodies. Blots were developed with the Western Lightening® Plus ECL kit (PerkinElmer). The primary antibodies including anti-Ppp2r1a (Genetex GTX-102206, 1:500), anti-phospho Tyr307 PP2AC (Santa Cruz sc-271903, 1:100), anti-Runx2 (Cell Signaling 8486, 1:1000), anti-human phospho Ser451 Runx2 (Bioss bs-5685, 1:300), anti-PPARγ (ABclonal A0270, 1:500), anti-phospho Ser492 BRD4 (Millipore ABE1451, 1:500), anti-Osterix (Bioss bs-1110, 1:300), anti-collagen X (Abcam ab58632, 1:100), anti-MMP13 (Genetex GTX-100665, 1:500) and anti-GAPDH (Genetex GTX-100118, 1:5000) were used. Immunoprecipitation was performed by Capturem™ IP & Co-IP Kit (Takara, 635721) and the results were analyzed with blotting the elution by anti- Ppp2r1a and anti- human phospho Ser451 Runx2.

The tissue sections were dewaxed in xylene solution, and then 3% hydrogen peroxide was used to inhibit the activity of endogenous peroxidase. The slices were incubated with 0.4% pepsin (Sigma-Aldrich) in 1 mM hydrochloric acid at 37°C for 1 h for antigen repair. The block solution was the PBS solution containing 5% BSA. After blocking with block solution at 37°C for 30 min, the slices were incubated with the primary antibodies against MMP13 (1:1000, GTX100665, GeneTex, CA, USA), CollagenII (1:1000, ab34712, Abcam, Cambridge, UK), TLR2 (1:1000, #29071, SAB, Nanjing, China) at 4°C overnight, and finally with a HRP-conjugated secondary antibody at room temperature for 1 h. Then, the slices were washed with PBS and developed, and sealed after the re-staining with methyl green (0.2%). The positive cells were counted by ImageJ.