OVX-BMSCs or HUVECs were seeded in 6-well plates (2×105 cells/well). OVX-BMSCs were cultured in medium supplemented with 100μM CoCl2 and different concentrations of SrR (0, 0.125, 0.25, 0.5, 1.0 and 2.0mM) for 48h. HUVECs were cultured in medium with SrR at 0.25mM for 0, 15, 30, 60 and 90 min. Then cells were washed by cold PBS, and lysed with RIPA for protein extraction. Total protein concentration was measured by a BCA protein assay kit.
Equal amount of protein samples were separated by 12% SDS-PAGE, and transferred to a polyvinylidene fluoride membrane. After blocking with 5% skim milk for 1h, the membrane was incubated with mouse anti-rat HIF-1α (1:1000, CST, USA), SDF-1(1:200, Abcam, UK), VEGF (1:1000, Abcam, UK) and β-actin (1:5000, Sigma, USA) for OVX-BMSCs, rabbit anti human AKT, phosphorylated-AKT (p-AKT), mTOR, phosphorylated -mTOR (p-mTOR) (1:1000, CST, USA) for HUVECs at 4 °C overnight. Then followed by a 2h incubation with HRP-conjugated goat anti-rabbit or rabbit anti mouse IgG (1:3000, Beyotime, China) at room temperature and washed by PBST for 3 times. Immunoreactive bands were detected by enhanced chemiluminescence (ECL) reagent (Pierce, USA), visualized by autoradiography and quantified by the Quantity One analysis system (Bio-Rad, USA). β-actin served as the internal control.
Equal amount of protein samples were separated by 12% SDS-PAGE, and transferred to a polyvinylidene fluoride membrane. After blocking with 5% skim milk for 1h, the membrane was incubated with mouse anti-rat HIF-1α (1:1000, CST, USA), SDF-1(1:200, Abcam, UK), VEGF (1:1000, Abcam, UK) and β-actin (1:5000, Sigma, USA) for OVX-BMSCs, rabbit anti human AKT, phosphorylated-AKT (p-AKT), mTOR, phosphorylated -mTOR (p-mTOR) (1:1000, CST, USA) for HUVECs at 4 °C overnight. Then followed by a 2h incubation with HRP-conjugated goat anti-rabbit or rabbit anti mouse IgG (1:3000, Beyotime, China) at room temperature and washed by PBST for 3 times. Immunoreactive bands were detected by enhanced chemiluminescence (ECL) reagent (Pierce, USA), visualized by autoradiography and quantified by the Quantity One analysis system (Bio-Rad, USA). β-actin served as the internal control.