Antimouse igg conjugated horseradish peroxidase

Total cell lysates extracted from the wild‐type or mutated POU4F3 plasmids transfected HEK 293 cells were subjected to sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and examined by Western blot with mouse monoclonal anti‐Flag antibodies (ZSGB‐Bio) and mouse monoclonal anti‐β‐actin antibodies (ZSGB‐Bio), and then followed by antimouse IgG conjugated horseradish peroxidase (ZSGB‐Bio). The signals were detected using the enhanced chemiluminescence system.

Total protein was extracted from the HEK293 cells transfected with the WT and mutant AIFM1 plasmids. The proteins were denatured and separated by 10% SDS-PAGE electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes (ISEQ00010, Merck Millipore, China). After blocking, the membranes were incubated with mouse monoclonal anti-GFP antibody (Proteintech, Wuhan, China) and mouse monoclonal anti-β-actin antibody (ZSGB-Bio, Beijing, China), and followed by anti-mouse IgG conjugated horseradish peroxidase (ZSGB-Bio, Beijing, China). Finally, the immunoblots were detected with an Immobilon Western HRP Substrate kit (WBKLS0100, Millipore, Schaffhausen, Switzerland) using the enhanced chemiluminescence system (Tanon5200, Shanghai, China). The gray scale of each band was quantified with ImageJ software and normalized by β-actin.