Immun star western chemiluminescence kit

Liver sections from mice were crushed in liquid nitrogen and then lysed in NP 40 lysis buffer (50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5 mM MgCl₂, 0.5% (v/v) Nonidet P-40, protease inhibitor cocktail, Beyotime Biotech, Beijing, China) on ice for 30 min. The protein concentration was determined using a BCA Protein Assay Kit (Beyotime Biotech, Beijing, China) following the manufacturer’s procedure. Samples were run on a 10% SDS polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with primary antibodies Foxp3 (1:2000 dilutions, proteintech, Chicago, USA), RORγt (1:2000 dilutions, proteintech, Chicago, USA). After incubation with secondary antibodies (1:2000, Sigma, San Diego, CA, USA), the signal was detected using the Immun-Star Western Chemiluminescence kit (BioRad, Hercules, CA, USA), quantified using the ChemiDoc XRS Imager and analyzed with the software Image Lab from Bio-Rad (BioRrad, Hercules, CA, USA). Tublin was used as endogenous control and the data were normalized by Tublin levels.