Enhanced ecl chemiluminescent substrate kit

The protein levels of TNFR1, JNK, p-JNK, PFKFB3, and α-SMA were detected by western blot. The cells were lysed on ice for 15 min in RIPA lysis buffer (Beyotime, China) containing 1% phenyl methyl sulfonyl fluoride (PMSF), protease inhibitor cocktail, and phosphatase inhibitor cocktail (KangChen, China). After centrifugation, the supernatants were collected and the protein quantification was determined using the BCA assay kit (Thermo scientific, USA). The proteins were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, Germany). Then, the membranes were incubated with appropriate primary and secondary antibodies, respectively. Subsequently, the immunoblots were detected by Image Lab TM software (Bio-Rad, USA) using Enhanced ECL Chemiluminescent Substrate Kit (Vazyme, China).