Horseradish peroxidas

Cells grown in 24-well plates were lysed on ice using 200 μL RIPA buffer (Beyotime Biotechnology, Shanghai, China) for 30 min. Next, the lysis mixtures were centrifuged and the supernatants were collected. BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA) was used for quantification of proteins. Then, proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and electrophoretically transferred to PVDF membranes. Afterwards, membranes were blocked in 5% bovine serum albumin (BSA) for 2 h at room temperature and then probed with primary antibodies at 4 °C overnight. The primary antibodies used in this study include anti-GAPDH (ab181602, Abcam, Cambridge, UK), anti-RIP2 (70R-10459, fitzgerald), anti-IL1β (abx132185, Abbexa, Cambridge, UK), anti-IL8 (abx100965, Abbexa), anti-IL6 (abx177189, Abbexa), anti-NFκB p65 (10745-1-AP, Proteintech, Wuhan, China), and anti-IκBα (10268-1-AP, Proteintech). The primary antibodies were used at a dilution of 1:1000. Then, the membranes were incubated with secondary antibodies marked by horseradish peroxidas (Sigma-Aldrich, St. Louis, MI, USA) at a 1:10,000 dilution at room temperature for 2 h. Immunoblots were visualized by enhanced chemiluminescence (ECL kit, Santa Cruz Biotechnology, Dallas, TX, USA). The images were analyzed using Image Lab™ Software (Bio-Rad, Hercules, CA, USA).

Cells from each of the four groups were lysed on ice using 200 μL RIPA buffer (Beyotime Biotechnology, Shanghai, China) for 30 min. Next, the lysis mixtures were centrifuged and the supernatants were collected. BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA) was used for quantification of proteins. Then, the isolated proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and electrophoretically transferred to PVDF membranes. Afterwards, membranes were blocked in 5% BSA for 2 h at room temperature and then probed with the primary antibodies at 4 °C overnight. The primery antibodies included anti-GAPDH (ab181602, Abcam, MA, USA), anti-RIP2 (70R-10,459, Fitzgerald, MA, USA), anti-HSP90AB1 (CBDH1187, Creative Biolabs, NY, USA), anti-BID (AB10002, MilliporeSigma, ON, Canada), and anti-CASP9 (STJ96979, St John’s Laboratory, London, UK) were used at a dilution of 1:1000. Then the membranes were incubated with secondary antibodies tagged with horseradish peroxidas (Sigma-Aldrich, St. Louis, MI, USA) at a 1:10000 dilution at room temperature for 2 h. Then, immunoblots were visualized by enhanced chemiluminescence (ECL kit, Santa Cruz Biotechnology, Dallas, TX, USA). The blots were visualized by using Image Lab™ Software (Bio-Rad, Hercules, CA, USA).