Bicinchoninic acid assay kit

Total proteins were extracted from SOSP-9607 cells using radioimmunoprecipitation assay lysis buffer (Bioswamp) and quantified using a bicinchoninic acid assay kit (Bioswamp). Each sample containing 20 μg of protein was separated and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). The membranes were blocked using 5% skim milk and incubated with primary antibodies against glucose transporter 1 (GLUT1, MAB37348, 1 : 1000), hexokinase 1 (HK1, MAB37234, 1 : 1000), lactate dehydrogenase A (LDHA, PAB30703, 1 : 1000), vimentin (PAB40646, 1 : 1000), E-cadherin (PAB43792, 1 : 1000), PI3K (PAB30084, 1 : 1000 dilution), p-PI3K (PAB43641-P, 1 : 1000), Akt (PAB30596, 1 : 1000 dilution), p-Akt (PAB43298-P, 1 : 1000), mTOR (PAB30674, 1 : 1000 dilution), p-mTOR (PAB36313-P, 1 : 1000), or GAPDH (PAB36269, 1 : 1000) for 1 h at room temperature, followed by incubation with goat anti-rabbit IgG (SAB43714, 1 : 20 000) secondary antibodies for 1 h at room temperature. All antibodies were supplied by Bioswamp. GAPDH served as an internal reference.

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.