Goat anti rabbit igg secondary antibody

Whole proteins were extracted from KGN cells using radioimmunoprecipitation assay lysis buffer (Solarbio), and the protein concentration was quantified using a bicinchoninic acid assay kit (Solarbio). In brief, 20 μg of proteins in each group was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were then blocked with 5% skim milk and cultured for 1 h with primary antibodies against TSC1 (Bioswamp, China), mTORC1 (Bioswamp, China), and phosphorylated (p)-mTORC1 (Abcam. USA), AKT (Bioswamp, China), p-AKT (Cell Signaling Technology, USA), and GAPDH (housekeeping control, Bioswamp, China), followed by 1 h of incubation with goat anti-rabbit IgG secondary antibody (Bioswamp, China).

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.