Rabbit polyclonal anti hsp90

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 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-Pten (1:1000 dilution, Cell Signaling), rabbit polyclonal anti-p16 (1:1000 dilution, clone M156; Santa Cruz Biotechnology), rabbit monoclonal anti-TNFR1 (1:1000 dilution, Cell Signaling), rabbit polyclonal anti-HSP90 (1:1000 dilution, Cell Signaling), mouse monoclonal anti-α-Tubulin (1:1000 dilution, Cell Signaling), rabbit monoclonal anti-STAT6 (1:1000 dilution, Cell Signaling), goat polyclonal anti-pSTAT6 (1:500 dilution, Santa Cruz Biotechnology). 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).