Rapid gel preparation kit

Human HCC samples were obtained via surgical resection of HCC patients from The Second Affiliated Hospital of Chongqing Medical University. The protocols were reviewed and supported by the Ethics Committee of The Second Affiliated Hospital of Chongqing Medical University Approval Number (2020): Institutional Review Board (IRB) (STUDY) No. 88, Chongqing, China. Tissue lysis was performed using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). Protein was extracted from tissues by centrifugation at 12,000g and detected by BCA assays at 562 nm. A rapid gel preparation kit (Beyotime Biotechnology, Shanghai, China) was used to configure the gel distribution at a 140-V constant voltage and 230 mA constant current electroconversion. Primary antibody was incubated at 4°C overnight. Secondary antibody was incubated at 37°C for 1 h. Phosphate-buffered saline was used to wash test strips three times for 10 min each. High-sensitivity ECL (Bio-Rad, USA) solution was used for the exposure strip. ImageJ software was used to analyze the results. RNA extraction and qPCR were performed using RNAiso reagent and a Prime Script™ Reverse Transcriptase kit (TaKaRa, Shiga, Japan) following the manufacturer’s instructions. The specific antibodies and primers are detailed in the supplementary materials.

RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) was used for cell and tissue lysis. Protein was extracted by centrifugation at 12,000 g and detected by BCA assay at 562 nm. A rapid gel preparation kit (Beyotime Biotechnology, Shanghai, China) was used to configure the gel distribution at 130 V constant voltages and 240 mA constant current elec-troconversion. Primary antibody was incubated at 4 °C overnight. Secondary antibody was incubated at 37 °C for 1 hour. Phosphate buffered saline was used to wash test strips three times for five minutes each. High-sensitivity ECL (Bio-Rad, USA) solution was used for the exposure strip. ImageJ software was used to analyze results. RNA extraction and qPCR were performed using RNAiso reagent and a PrimeScript™ Reverse Transcriptase kit (Takara, Shiga, Japan) following manufacturer instructions (Par. 1-7).