Rabbit anti gapdh antibody

Cells were lysed in lysis buffer (RIPA buffer, 1 mM PMSF, Phosphatase inhibitor Cocktail and protease inhibitor cocktail) on ice for 15 min. Bone tissues were ground with a mortar in liquid nitrogen and were lysed in lysis buffer at 4 ˚C for 30 min. Protein fractions were collected by centrifugation at 12,000 × g, 4 ˚C for 10 min and then 10 mg of lysates were subjected to SDS-PAGE and transferred to nitrocellulose filter membrane (NC) membranes. The membranes were blocked with 5% skimmed milk and incubated with specific antibodies overnight. The antibody of used were listed: Rabbit anti-NFATc1 (1:1000, abclonal, A1539), rabbit anti-Ano1 (1:1000, abclonal, A10498), rabbit anti-p-CaMKIV antibody (1:1000, ImmunoWay, YP0043), rabbit anti-CaMKIV antibody (1:1000, Cell Signaling Technology, 4032), rabbit anti-Creb antibody (1:1000, Cell Signaling Technology, 9197), rabbit anti-p-Creb antibody (1:1000, Cell Signaling Technology, 9198), rabbit anti-Gapdh antibody (1:5000, Abways, AB0036).

BMSCs or primary osteoblasts were lysed in buffer (150 mmol·L⁻¹ NaCl, 50 mmol·L⁻¹ Tris, pH 7.8, 10% glycerol, 1 mmol·L⁻¹ EGTA, 0.5% NP-40, 1 mmol·L⁻¹ EDTA, 1 mmol·L⁻¹ PMSF, 1x cocktail) on ice with vigorous shaking for 30 min. The lysates were subjected to Western blotting with the indicated antibody. The following antibodies were used in the study: rabbit anti-HuR antibody (1:1 000, Cell Signaling Technology, Cat. No. 12582, monoclonal), rabbit anti-Atf4 antibody (1:1 000, Cell Signaling Technology, Cat. No. 11815, monoclonal), and rabbit anti-Gapdh antibody (1:5 000, Abways, Cat. No. AB0036, monoclonal).