Figure 2

Heat-induced oligomerization and phosphorylation of HSF1. (A) Native anti-HSF1 blot showing heat-induced oligomerization of endogenous HSF1 in response to heat treatment at 44°C (HS) for the times indicated on top of the blot. T: HSF1 trimers; D: heterodimers; M: monomers. (B) Parallel cultures to those used in A for an analysis of HSF1 oligomerization were employed here for an examination of global or Ser³²⁶-specific phosphorylation of endogenous HSF1. The anti-HSF1 western blot shown reports the distribution of HSF1 forms of different apparent size (reflecting different levels of phosphorylation) in extract samples (lysate) or in protein immunoprecipitated from the same extracts by pSer³²⁶ antibody (ip). The portion of the blot depicted only shows protein signals larger than about 70 kD. (C) Detection of heat-induced phosphorylation of Ser³²⁶ by western blot using anti-pSer³²⁶ antibody. Parallel cultures were transfected with small amounts of FLAG-HSF1. One day later, the cultures were either left untreated (C) or were heat-treated for 30 min at 44°C (HS) and were processed for western blot immediately following the heat treatment. The anti-pSer³²⁶ blot reports on induction of Ser³²⁶ phosphorylation by heat, and the parallel anti-FLAG blot shows that similar amounts of FLAG-HSF1 were compared in the anti-pSer³²⁶ blot. Data from densitometry are shown below the blots. Brackets shown on the side of blots indicate lengths of regions scanned. In A, the monomer signal was quantitated.