Horseradish peroxidase conjugated goat anti rabbit or anti mouse igg sc 2004 or sc 2005

Proteins were extracted using an ice-cold RIPA lysis buffer (P00138, Beyotime, Nanjing, China) with proteinase inhibitor (ST506, Beyotime, Nanjing, China). Equal amounts of proteins were measured using an BCA protein assay kit (A045-3, Jiancheng, Nanjing, China). Proteins were separated by electrophoresis and then electronically transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, USA) using a BioRad system (BioRad, Hercules, USA). The membrane was blocked in 5% skimmed milk at room temperature for 2 h and subsequently incubated overnight at 4°C with corresponding primary antibodies, rabbit anti-BCL2 (1:200), mouse anti-GRP78 (1:200), rabbit anti-LC3β (1:100, sc-28266, Santa Cruz Biotechnology, Santa Cruz, USA), rabbit anti-CYP11A1 (1:200, CSB-PA006389LA01HU, CusAb, Wuhan, China), mouse anti-BAX (1:100, ab5714) and mouse anti-β-actin (1:1000, ab8226, Abcam, Cambridge, UK). Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (sc-2004 or sc-2005, Santa Cruz Biotechnology, Dallas, USA) were then used to detect proteins using Clarity ECL Western Blot Substrate kits (BioRad, Hercules, USA) and exposed using a ChemiScope 3400 Mini machine (Clinx, Shanghai, China). Protein quantification was performed using densitometry analyses on Quantity One Software.