Radioimmunoprecipitation ripa buffer

Cells were harvested using cell dissociation buffer, centrifuged and washed with PBS prior to lysis using Radioimmunoprecipitation (RIPA) buffer (Sigma) containing protease inhibitor cocktail tablets (1 tablet per 10 ml RIPA buffer) (Roche Applied Science) at a concentration of 2 × 10⁷ cell/ml. Lysates were separated by SDS-PAGE and electrotransferred onto nitrocellulose membrane as previously described24 (link). The membrane was probed using the appropriate primary antibody with subsequent incubation with HRP-conjugated secondary antibody. The membrane was exposed to an autoradiographic film (Hyperfilm ECL, GE Healthcare, Amersham, UK) for the appropriate time period in an X-ray cassette before the film was manually developed in a dark room using Kodak GBX developer and fixer solutions (Sigma, Dorset, UK). Primary antibodies: anti-α-tubulin, 1:2000 dilution (T9026, Sigma, Dorset, UK); anti-AKT, 1:200 dilution (#9272, Cell Signalling Technology, MA, USA); anti-pAKT (Ser473), 1:200 (#4060S, Cell Signalling Technology, MA, USA); anti-STAT3, 1:200 dilution (SC-482, Santa Cruz Biotechnology Inc, TX, USA); anti-pSTAT3, 1:200 dilution (#9138S, Cell Signalling Technology, MA, USA).

Proteins from lysates of BMDMs were extracted in Radio-Immunoprecipitation (RIPA) Buffer (Sigma Aldrich) containing Halt^TM protease and phosphatase inhibitor cocktail (Fisher Scientific, Napean, ON, Canada). Immunoblots were prepared with Bolt^® Bis-Tris Plus Gel (ThermoFisher), and western blot analysis was carried out according to standard protocols, with antibodies specific for rabbit polyclonal raised against human NLRP3 (1:6500, sc-66846, Santa Cruz Biotechnology Inc., Santa Cruz, CA). GAPDH was used as a loading control (AM4300, ThermoFisher). Relative protein levels were normalized to GAPDH as determined by densitometry using Image J software (1.8v, NIH).