Odyssey color infrared laser scan imaging instrument

After treated with different stimulus, the cells were collected and lysed in ice-cold radio immunoprecipitation assay (RIPA) (Beyotime, China) buffer containing protease inhibitors phenylmethylsulfonyl fluoride (PMSF) (Beyotime, China) and protease inhibitor cocktail (Beyotime, China) and then centrifuged at 12000 rpm at 4°C for 15 min to obtain supernatants. Equal amount of protein lysates were loaded into a 5%-10-15% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were blocked in 5% nonfat milk for 1 h at room temperature and then incubated overnight with primary antibodies against NF-κB p65 (1 : 1000, CST, USA), NLRP3 (1 : 200, Novus, USA), ASC (1 : 200, Santa Cruz, USA), caspase-1 (1 : 200, Santa Cruz, USA), IL-1β (1 : 1000, Abcam, UK), IL-18 (1 : 1000, Abcam, UK), and GAPDH (1 : 1000, CST, USA). The membranes were subsequently incubated with fluorescent secondary antibody (1 : 15000, CST, USA) for 1 h at room temperature. Then, the membranes were washed again with TBST for 3 times, 5 minutes of each time. The protein bands were detected with an Odyssey color infrared laser scan-imaging instrument (LI-COR, USA). The images were analyzed using Odyssey Application Software 3.0.

DHA (Beijing Zhongsha Jinqiao Biotechnology Co., Ltd); SSeCKS siRNA (Santa Cruz, CA, USA); ELISA kit and TUNEL kit (Jiancheng, Nanjing, China); Perkin Elmer Microplate reader (PerkinElmer Victor 1420, USA); Ang-1, Ang-2, and VEGF mouse anti-human monoclonal antibodies (R&D Systems, Minneapolis, USA); SSeCKS rabbit anti-human monoclonal antibodies (Santa Cruz, CA, USA); Goat anti-mouse polyclonal antibodies and mouse anti-rabbit polyclonal antibodies (R&D Systems, Minneapolis, USA); Odyssey color infrared laser scan-imaging instrument (Li-Cor, USA). FACSC calibur flow cytometer (BD, New Jersey, USA).