Western Blot Analysis of Stress Proteins

Cell pellets were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 12 mM deoxycholic acid, 0.5% Nonidet P-40, and protease inhibitors). Protein samples were used for SDS-PAGE followed by Western blotting as previously described [30 (link)]. Nitrocellulose membranes were stained with primary antibodies against Tubulin (1 : 1000), p53 (1 : 1000), HSF1 (1 : 1000), Hsp27 (1 : 1000), Hsp90 (1 : 1000), and p-p38 (Santa Cruz Biotechnology, Dallas, TX). The nitrocellulose membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Bio-Rad), and immunoreactive bands were detected by a Fluorchem Imaging System upon staining with the ECL Select Western Blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, USA; RPN2235). The Western blots reported are from one experiment out of three different experiments that gave similar results.
Protein content was assayed by the method described by Lowry et al. [31 (link)].