Enhanced chemiluminescence reagent

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.

Each expression was detected with reference to existing work [18 (link)]. The harvested cells were extracted by means of radioimmunoprecipitation assay buffer (Sigma-Aldrich Co.) and then quantified by means of a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were incubated with primary antibodies (PARP, caspase-3, PD-L1, phosphorylated signal transducer and activator of transcription 1 (pSTAT1), STAT1, actin, and GAPDH; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). An enhanced chemiluminescence reagent (DoGenBio, Seoul, Republic of Korea) was used for signal development.