Sds page condensed electrophoresis

Cell total proteins were split using RIPA lysis buffer supplemented with 1 mmol/l phenylmethanesulfonyl fluoride (Beyotime) and separated with SDS-PAGE condensed electrophoresis (Beyotime). Polyvinylidene fluoride membrane (Beyotime) was cut off according to a size of gel, arrange gel-membrane sandwich and use it in electrotransfer. Next, the membranes were blocked in 5% BSA for 1 hour at room temperature. Then, primary antibodies against raptor (1 : 1000; Cell Signaling Technology, USA), rictor (1 : 1000; Cell Signaling Technology), VE-cadherin (1 : 1000; abcam, USA), Caspase-3 (1 : 1000; Cell Signaling Technology), Cleaved-Caspase-3 (1 : 1000; Cell Signaling Technology), phosphorylation or total Akt (Ser473) (1 : 1000; Cell Signaling Technology), mTOR (Ser2448) (1 : 1000; Cell Signaling Technology), p70 S6 kinase (p70S6K; Thr389) (1 : 1000; Cell Signaling Technology), PKC-a (Ser657) (1 : 1000; Cell Signaling Technology), and β-Actin (1 : 1000; Cell Signaling Technology) were applied with appropriate dilution at 4°C overnight. Peroxidase-conjugated secondary antibody (1 : 3000; Fcmacs) was incubated on the membrane for 1 h at room temperature. Finally, ECL detection was used and membranes were exposed with a chemiluminescence imaging system (Bioshine ChemiQ 4800mini, Ouxiang, Shanghai, China).

RIPA lysis buffer supplemented with 1 mmol/l phenylmethanesulfonyl fluoride (Beyotime) were used to extract total proteins of MSCs. Extract total proteins of MSCs were separated with SDS-PAGE condensed electrophoresis (Beyotime) and transferred onto polyvinylidene fluoride membranes (Beyotime). Then, we blocked the membranes in 5% BSA and dyed them for 1 h at room temperature and dyed at 4 °C overnight with primary antibodies against β-Actin (1:1000; Cell Signaling, USA), HIF-1α (1:1000;Cell Signaling), Hyp564 HIF-1α (1:1000;Cell Signaling), CXCR4 (1:100; Abcam, USA), CXCR7(1:1000;Abcam), Caspase-3 (1:1000;Cell Signaling), and Cleaved-Caspase-3 (1:1000;Cell Signaling). Peroxidase-conjugated secondary antibody (1:3000; Fcmacs) was used to incubate the membrane at room temperature for 1 h. Finally, ECL was applied to detect the bands with a chemiluminescence imaging system (Bioshine ChemiQ 4800mini, Ouxiang, Shanghai, China).