Enhanced chemiluminescence substrate buffer

The expression of apoptosis-related proteins caspase 3, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated x (Bax), and cytochrome C (Cyt-c) was detected using Western blot assay. Total protein content was extracted using radioimmunoprecipitation assay lysis buffer (Bioswamp) supplemented with protease and phosphatase inhibitors. A bicinchoninic acid kit (Bioswamp) was used for protein quantification. Proteins (20 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking with 5% skim milk, the membranes were incubated with primary antibodies against caspase 3 (Abcam, ab13847, 1:1000), cleaved caspase 3 (Abcam, ab2302, 1:1000), Bcl-2 (Abcam, ab196495, 1:1000), Bax (Abcam, ab182733, 1:2000), Cyt-c (Abcam, ab133504, 1:5000), and GAPDH (CST, 2118, 1:1000) overnight at 4°C, followed by incubation with goat anti-rabbit immunoglobulin (Ig)G secondary antibody (Bioswamp, PAB150011, 1:10000) for 1 h at room temperature. Immunoreactivity was visualized by colorimetric reaction using an enhanced chemiluminescence substrate buffer (Millipore, MA, USA). The membranes were then detected using a Tanon-5200 apparatus (Tanon Science & Technology Co., Ltd., Shanghai, China) and the band gray values were read.

Total proteins were extracted from EPCs using radioimmunoprecipitation assay lysis buffer (Bioswamp) supplemented with protease and phosphatase inhibitors. The proteins were quantified using a bicinchoninic acid assay kit (Bioswamp). The obtained proteins (20 μL) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% skim milk for 2 h at room temperature and incubated overnight at 4°C with the following primary antibodies: PI3K (Abcam, 1:1000), Akt (Bioswamp, 1:1000), p-Akt (Bioswamp, 1:1000), glycogen synthase kinase 3β (GSK3β, Abcam, 1:5000), p-GSK3β (Abcam, 1:1000), extracellular signal-regulated kinase 1/2 (ERK1/2, Abcam, 1:1000), p-ERK1/2 (Abcam, 1:1000); caspase 3 (Bioswamp, 1:1000), angiopoietin (Ang)1 (Abcam, 1:500), Ang 2 (Abcam, 1:5000), and glyceraldehyde 3-phosphate dehydrogenase [GAPDH] (CST, 1:1000). After washing, the membranes were incubated with a goat anti-rabbit IgG secondary antibody (Bioswamp, 1:20000) at room temperature for 1 h. Immunoreactivity was visualized by colorimetric reaction using enhanced chemiluminescence substrate buffer (Millipore) using an automatic chemiluminescence analyzer (Tanon-5200, Shanghai, China). The band gray values were measured by TANON GIS software.