Il23r

Equal amounts of protein (20 μg) extracted from Th17 cells using RIPA (Biyuntian, Shanghai, China) were electrophoresed using 10% SDS/PAGE, transferred to Polyvinylidene fluoride membranes, blocked, and then incubated at 4 °C overnight with the following primary antibodies: SGK1 (1 : 500; Abcam, ab43606), p‐Foxo1 (1 : 500; Abclonal, Boston, MA, USA, AP0176), and IL‐23R (1 : 500; Abcam, ab175072). The blots were then incubated with an HRP‐conjugated secondary antibody (1 : 2500; Proteintech) at room temperature for 1 h and visualized using a Bio‐Rad Gel Doc EZ Imaging System (Bio‐Rad, Hercules, CA, USA) with enhanced chemiluminescence reagents (Millipore, Boston, MA, USA). All western blot analyses were performed at least three times, and the protein band densities were quantified using imagej software (NIH, National Institutes of Health, Bethesda, MA, USA).

Total proteins were extracted from PBMCs using RIPA lysis buffer (Solarbio, China), and protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA). An equal amount of protein was loaded onto 10% SDS-PAGE and subjected to electrophoresis, then transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, USA). The membrane was washed with Tris-buffered saline-Tween (TBST) (Solarbio, China), and blocked with 5% skim milk powder or bovine serum albumin (BSA) (Sigma-Aldrich, Germany). Incubation was carried out using specific primary antibodies against STAT3 (Abcam, U.K), pSTAT3 Y705 (Abcam, U.K), ADAM17 (R&D Systems, USA), IL-23 R (Abcam, U.K), and GAPDH (Santa Cruz, USA), followed by the incubation with the secondary antibodies HRP Goat anti-Mouse antibody (abclonal, USA) or HRP Goat Anti-Rabbit IgG (abclonal, USA). Pierce™ ECL Western Blotting Substrate (Thermo Fisher, USA) was used as the chemiluminescent substrate, and X-ray imaging was used to image the bands. Relative expression levels of proteins were quantified by densitometric values of fluorogram bands which was analyzed by Image J software (NIH, USA) and each value was normalized to those corresponding control protein band.