Trans blot electrophoretic transfer kit

Total cellular protein was extracted using the cell lysis buffer (Beyotime), and concentrations were determined by BCA protein assay kit (Beyotime). Lysates were loaded in SDS-AGE gel and electrophoresed. Proteins were transferred from gel to nitrocellulose membrane using a trans-blot electrophoretic transfer kit (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked in 5% skim milk in TBST buffer and incubated with primary antibodies anti-JMJD6 (1:3000; H-7, Santa Cruz Biotechnologies, Dallas, TX, USA), anti-histone H4R3me2a (1:3000; active motif), anti-HA tag (1:4000; HA.C5, Abcam), anti-α-tubulin (1:6000, DM1A, CST), anti-PDE1C (1:2000; Abcam) and anti-GAPDH (1:4000; 6C5, Abcam). After washing, the membranes were incubated with HRP goat anti-mouse IgG (Beyotime) or HRP goat anti-rabbit IgG (Beyotime). Membranes were then incubated in BeyoECL plus (Beyotime) and then imaged using Chemidoc imaging system (Bio-Rad Laboratories). Gray value was analyzed using Image J (Java 1.8.0).

After 21 days osteogenic differentiation of AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs, the total cellular protein was extracted using the cell lysis buffer (Beyotime), and concentrations were determined by Bradford protein assay kit (Bio-Rad). Proteins were loaded in SDS-AGE gel and electrophoresed at 80 V for 30 min and 140 V for 60 min. Then, proteins were transferred from gel to nitrocellulose membrane using a trans-blot electrophoretic transfer kit (Bio-Rad). Membranes were blocked in 5% skim milk in TBST buffer for 60 min and incubated with primary antibodies osterix (1:3000, ab94744, abcam 45kd), collagen I (1:3000, ab34710, abcam 125kd), osteopontin (1:2000, ab166709, abcam 65kd), osteocalcin (1:2000, ab93876, abcam 11kd), Tubulin(1:4000, ab4074, abcam 50kd), phosphate-Akt (1:2000, 193H12, Cell signaling technology), Total-Akt (1:2000, C67E7, Cell Signaling Technology), phosphate-Erk1/2 (Thr202/Tyr204, Cell Signaling Technology), total- ERK1/2 (1:2000, Cell Signaling Technology). After washing with TBST buffer, the membranes were incubated with HRP goat anti-mouse IgG (1:3000, Beyotime) or HRP goat anti-rabbit IgG (1:3000, Beyotime). Membranes were then incubated with pierce ECL western blotting substrate (Thermo fisher) and then imaged using chemidoc imaging system (Bio-Rad).