Peroxidase conjugated secondary antibodies

Total proteins were collected from cells using RIPA lysis buffer (Solarbio, Beijing, China) with protease inhibitors (Solarbio, Beijing, China) on ice and quantified by a bicinchoninic acid (BCA) assay (Solarbio, Beijing, China). Equal amounts of protein were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Solarbio, Beijing, China). The protein bands were blocked in freshly prepared 5% skim milk, followed by blotting with antibodies against Bcl-2 (1 : 1000; Abcam, Cambridge, UK), Bax (1 : 1000; Abcam, Cambridge, UK), caspase 3 (1 : 1000; Cell Signaling Technology, USA), cleaved-caspase 3 (1 : 1000; Cell Signaling Technology, USA), caspase 9 (1 : 1000; Cell Signaling Technology, USA) and GAPDH (internal reference gene, 1 : 1000; Elabscience Biotechnology Co., Ltd., China) at 4°C overnight. Then, peroxidase-conjugated secondary antibodies (1 : 5000; Elabscience Biotechnology Co., Ltd., China) were used to incubate the membranes. Subsequently, electrochemiluminescence (ECL) solution was applied to detect each band with a chemiluminescence imaging system. The gray value of the protein bands was measured by ImageJ software and normalized to the expression of GAPDH.