Rabbit polyclonal anti phospho stat3 tyr705

Tissue and cell lysates were prepared with RIPA buffer (1x PBS, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail; Roche). Total protein concentration was measured using BCA Protein Assay Kit (Cat: 23225; Pierce, Rockford). Equal amounts of proteins were separated by SDS-PAGE and western blotted onto a 0.45 μm-nitrocellulose membrane. Membranes were blocked in 5% defatted milk or 5% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST), probed with diluted antibodies and incubated at 4°C overnight. The following primary antibodies were utilized: rabbit polyclonal anti-HSP90 (1:1000 dilution, Cell Signaling), rabbit polyclonal anti-phospho-Stat3 (Tyr705) (1:1000 dilution, Cell Signaling), rat monoclonal anti-RORγt (5:1000 dilution, clone AFKJS-9, eBioscence), rabbit polyclonal anti-IL23R (H-300) (1:1000 dilution, Santa Cruz). After washing in TBST, the membrane was incubated with secondary antibody conjugated with horseradish peroxidase (HRP) (dilution 1:5000, Cell Signaling). The protein bands were visualized using the ECL Western Blotting Substrate (Pierce).