Anti alp

Cells were lysed by RIPA buffer supplemented with protease inhibitor. The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated with primary antibodies, then with horseradish peroxidase (HRP)-conjugated secondary antibodies, and visualized by chemiluminescence reagent (Proteintech, Wuhan, Shanghai). The primary antibodies used included antibodies produced by Abcam (Cambridge, MA, USA): anti-osterix, anti-ALP, and anti-osteopontin (OPN); antibodies by Cell Signaling Technology (Danvers, MA, USA): anti-PPARγ, anti-C/EBPα, anti-phospho-src homology 2 domain-containing transforming protein C1 (SHC1) (Tyr317), anti-ERK1/2, and anti-phospho-ERK1/2 (Thr202/Tyr204); antibody by ABclonal (Wuhan, China): anti-nuclear factor of activated T cells 1 (NFATC1); antibody by Beyotime (Shanghai, China): anti-cathepsin K (CTSK); antibodies by Proteintech (Wuhan, China): anti-OSMR, anti-fatty acid-binding protein 4 (FABP4), anti-microtubule-associated protein 1 light chain 3 (LC3), anti-autophagy-related gene 5 (ATG5), anti-autophagy-related gene 7 (ATG7), anti-Beclin 1, anti-P62, anti-SHC1, and anti-β-actin. The expression levels of the target genes were analyzed by dividing the grayscale of the target bands with that of β-actin.

Total proteins were extracted with lysis buffer (10 mM Tris-HCL, 1 mM EDTA, 1% sodium dodecyl sulfate, 1% Nonidet P-40, 1:100 proteinase inhibitor cocktail, 50 mM b-glycerophosphate, and 50 mM sodium fluoride). Aliquots of 20–60 mg per sample were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to the polyvinylidene fluoride membranes and blocked with 5% nonfat milk powder in PBST (PBS with 0.1% Tween). Next, they were incubated with the following primary antibodies overnight: anti-Osterix, anti-Runx2, anti-ALP, anti-P65, anti-p-P65, anti-IKBα, anti-IKK, anti-p-IKK (Cell Signaling Technology). The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Boster, Wuhan, China). The blots were visualized using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s recommended instructions.