Spa 901

Cellular proteins were extracted with lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxicholate, 0.1% SDS, 1 mM dithiothreitol (DTT), 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride (PMSF)) supplemented with a protease inhibitor cocktail (Complete). In some experiments, whole cell extracts (43 (link),44 (link)) were used to assess levels of HSF1. Protein concentration in cell lysates was determined by a Bradford-based protein assay. A total of 25 μg of proteins were resolved by SDS-PAGE, transferred to a PVDF membrane and analyzed by immunoblotting using mouse anti-human HSP72 monoclonal antibody C92F3A-5, rabbit anti-human HSF1 polyclonal antibody SPA-901 or rat anti-mouse HSF2 monoclonal antibody SPA-960 (all from Enzo). A mouse anti-human GAPDH monoclonal antibody 9484 (Abcam) was used as a loading control. HSF1 oligomerization was assessed using amine-specific cross-linker ethylene glycol bis-succinimidyl succinate (EGS) (Pierce). Whole cell extract (50 μg) prepared as previously described (43 (link),44 (link)) was incubated with 0.5 mM EGS for 30 min at RT. The cross-linking reaction was quenched by the addition of 50 mM glycine/0.025 mM Tris, pH 7.5 and incubation for 15 min at RT. Proteins were fractionated through a 6% SDS-PAGE gel and analyzed by immunoblotting using anti-HSF1 antibody SPA-901.