Western blot stripping buffer

Whole cell lysis was carried out using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) according to the manufacturer’s protocol, with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Total protein was quantified with Pierce BCA^TM Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Protein extract was separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. Subsequently, the immobilized proteins were incubated with the appropriate primary antibody: cytochrome c (#11940), Smac/Diablo (#2954), HtrA2/Omi (#9745) and β-Tubulin (#2128) (Cell Signaling Technology, MA, USA). Finally, the appropriate secondary antibody conjugated with horseradish peroxidase (#7074, Cell Signaling Technology, MA, USA) was applied. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA). Western blot stripping buffer (Thermo Scientific, MA, USA) was used to remove the antibodies from the membrane.

The HEPG2 cells were preincubated with or without 50 µM of KC for 1 h and then treated with or without 0.5 mM of tBHP for 12 h. Then cellular proteins were extracted in radioimmunoprecipitation assay buffer (RIPA buffer). The protein concentration was measured using Bradford’s assay and 30 µg of protein from each sample was subjected to Western blot analysis. For the Western blot analysis, the protein was separated on an SDS-PAGE gel by electrophoresis and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were incubated with various primary antibodies overnight and after washing in tris-buffered saline with 1% Tween 20 (TBST) buffer, the membranes were further incubated with appropriate corresponding horseradish peroxidase-conjugated secondary antibodies. After further washing, the proteins on the membranes were visualized using an enhanced chemiluminescence reagent (Dogenbio). The membranes were stripped with Western blot stripping buffer (Thermo scientific) and incubated with actin antibodies and the immunoblotting procedure repeated. The protein bands were quantified using ImageJ analysis software and normalized to the expression of the internal control β-actin; the results were further normalized to the control.

Proteins were separated in a SDS-PAGE gel by electrophoresis and transferred onto polyvinylidene fluoride (PDVF) membrane33 (link). Membranes were blocked with TBST (TBS with 0.1% tween 20) containing 5% milk powder (blocking buffer) for 1 h at room temperature or overnight at 4°C. Membranes were immunoblotted with the anti-GFP mouse antibody (diluted 1:500 in blocking buffer) for 1 h at room temperature or overnight at 4°C. Membranes were washed and then incubated with the HRP-conjugated anti-mouse secondary antibody (diluted 1:2500 in blocking buffer) for 1 h at room temperature. Membranes were then incubated in ECL select substrate and bands visualised using the ChemiDocTM XRS system (Bio-Rad)34 (link)35 (link). Blots probed with the anti-GFP mouse antibody were stripped with western blot stripping buffer (Thermo Scientific) and reprobed with the anti-VSVG rabbit antibody (diluted 1:1000) in blocking buffer) and the HRP-conjugated anti-rabbit secondary antibody (diluted 1:2500 in blocking buffer) as described above.