2.7. Protein Isolation and Western Blot

Proteins were extracted from osteoblasts by RIPA lysis buffer (Solarbio, China) on ice, and the supernatant lysate was collected after centrifugation at 12,000 rpm for 15 min. Then, the concentration was measured using BCA Protein Assay Kit (Solarbio, China). After protein denaturation for 5 min at 100°C, the proteins were separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes by a wet transfer apparatus. Bands were blocked in 5% dried milk for 1 h and incubated overnight at 4°C with first antibodies against COLI (Cell Signaling Technology, CST, 84336, 1 : 1000, USA), ALP (Abcam, ab108337, 1 : 1000, UK), RUNX2 (CST, 8486, 1 : 1000, USA), PKG2 (Santa Cruz, sc-393126,1 : 500,USA), PDE5 (Abcam, ab14672,1 : 1000, UK), PLCβ1 (Abcam, ab182368, 1 : 1000, UK), IP3R (Santa Cruz, sc-377518,1 : 500,USA), PERK (Bioss, bs-2469R, 1 : 500, China), phospho-PERK (Bioss, bs-3330R, 1 : 500, China), GRP78 (Bioss, bs-1219R, 1 : 500, China), ATF4 (Bioss, bs-1531R, 1 : 500, China), CHOP (CST, 2895, 1 : 1000, USA), β-actin (Abways, AB0035, 1 : 5000 China), and GAPDH (Proteintech, 10494-1-AP, 1 : 5000, China). The next day, the membranes were washed and incubated with anti-rabbit or anti-mouse HRP-conjugated secondary antibodies. The protein bands were visualized under Amersham Imager 600 (Millipore, USA), and intensities were calculated by ImageJ software.