Western Blotting for Protein Expression Analysis

Radioimmunoprecipitation assay (RIPA) lysis reagent (Beyotime) was used for protein extraction, followed by protein quantification using the BCA kit (Beyotime). Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE; 10%) was performed to separate the proteins before transferring the protein bands onto polyvinylidene fluoride (PVDF) membranes. Subsequently, the membranes were blocked using a blocking buffer (Beyotime) for 30 min. They were then incubated overnight at 4°C with primary antibodies targeting MGMT (ab108630; Abcam, Cambridge, MA, USA; 1/2000 dilution) or GAPDH (ab9485; 1/2500 dilution). The membranes were then incubated with a secondary antibody (ab205718; Abcam; 1/5000 dilution) for 1.5 h at room temperature. Finally, the protein signals were visualized using the enhanced chemiluminescence (ECL) kit (Beyotime) [31 (link)].