Anti irs

Sample protein concentrations of cells or tissues were determined by BCA Protein Assay kit. Typically, total protein (40 μg) was electrophoresed by 8% SDS-PAGE and then transferred onto PVDF membranes. Primary antibodies used in this study were anti-NLRP3, anti-IRS (for phosphor-Ser³⁰⁷IRS), anti-IRS, anti-PI3K, anti-AKT (for phosphor-Thr³⁰⁸AKT), anti-AKT, and anti-Glut4 (all from Cell Signaling Technology, Danvers, MA, USA). After blocking, membranes were immunoblotted with primary and secondary antibodies, followed by detection with an ECL system.

Homogenization was performed using the lysis buffer (Beyotime, Wuhan, China). Bradford assay (Bio‐Rad, Roseville, CA, USA) was used for protein quantification. SDS/PAGE was used to study these proteins, which were isolated using 8–15% polyacrylamide gels (Bio‐Rad) and transferred to polyvinylidene difluoride membranes (Millipore, Franklin, MA, USA). After blocking, the membranes were incubated overnight (4 °C) in the presence of particular primary antibodies (anti‐IGFR, anti‐phospho‐IGFR, anti‐AKT, anti‐phospho‐AKT, anti‐AR, anti‐IRS, anti‐phospho‐IRS, anti‐β‐actin, and anti‐LKB1; Cell Signaling Technology, Franklin, MA, USA). Secondary antibodies were subsequently supplemented. Enhanced chemiluminescence plus detection reagent (Pierce, Braketown, IL, USA) was used to measure the bands, which were further evaluated using the Omega 16ic Imaging System (Ultra‐Lum, Roseville, CA, USA).