Cells were lysed with TGNET buffer [50 mM Tris HCl pH7.5, 5% Glycerol, 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100] containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche). Lysates were centrifuged at maximum speed for 15 minutes at 4 °C. Supernatants were subjected to BCA protein assay for normalization, and then 40 μg of protein were used as input lysate to confirm protein expression. From remaining lysates, 700 μg of protein were subjected to immunoprecipitation by addition of 40 μl of Anti-FLAG M2 affinity beads (Sigma-Aldrich, A2220) or HA affinity beads (clone HA-7, Sigma-Aldrich, A2095) followed by incubation with rotation for 2 hours at 4 °C. After centrifugation, beads were washed 4 times with TGNET buffer. The proteins were then eluted with 30 μl of 2X SDS sample buffer by boiling at 95 °C for 5 minutes. Subsequently, samples were subjected to SDS-PAGE followed by western blotting. The following antibodies were used for western blot: HSF1 (Santa Cruz, sc-13516), HSP90 (Enzo Life Science, ADI-SPA-835), HSP70 (Enzo Life Sciences, ADI-SPA-810), HA (Rockland, 600-401-384), and FLAG (Sigma-Aldrich, A8592).
Additional Methods may be found in Supplemental Information.
Additional Methods may be found in Supplemental Information.