Fluorescent based anti rabbit igg secondary antibody

Protein extraction and western blots were performed as previously described [26 (link)]. Membranes were probed with primary rabbit antibodies against stathmin, p53, beta-actin, cyclin D1, bcl-2 and GAPDH, purchased from Abcam; or with antibodies against cleaved-PARP, PARP, cyclin B1, cdc2 and p-cdc2 (Tyr15), purchased from Cell Signaling Technology, Danvers, MA; and finally with fluorescent-based anti-rabbit IgG secondary antibody (Fermentas, Vilnius, Lithuania). Immunoreactive bands were scanned and analyzed using Image J software (NIH, Bethesda, Maryland) and the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).

Tissue protein was extracted using the Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime Biotechnology, Nanjing, China). Equal amounts of protein were electrophoresed on 10% SDS-polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked in 5% fat-free milk with 0.1% Tween-20 for 1 h at room temperature, followed by incubation with different primary antibodies including NLRP3 (1:1000, Sigma, St. Louis, MO, USA), Caspase 1 (1:1000, CST, Danvers, MA, USA), IL-1β (1:1000, Abcam, Cambridge, UK), Caspase 3 (1:1000, CST), cleaved Caspase 3 (1:1000, CST), Bcl-2 (1:800, Proteintech, Rosemont, IL, USA), poly (ADP-ribose) polymerase (PARP) (1:1000, CST), superoxide dismutase (SOD) 2 (1:1000, Abcam), catalase (1:1000, Abcam) or β-actin (1:1000, Sigma) overnight at 4 °C. After washing the membranes for three times, the blots were incubated with fluorescent-based anti-rabbit IgG secondary antibody (1:1000, Fermentas, Vilnius, Lithuania) for 1 h at room temperature. Protein was visualized using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).