Fig. 6: HSF1-S419 phosphorylation maintains proteostasis capacity.

a TRRAP promotes cell survival. Cells were infected with adenovirus expressing shRNA for HSF1, TRRAP or SCR, and were heat-shocked at 45 °C for the indicated periods. Number of viable cells excluding trypan blue was counted. Extracts of cells were subjected to immunoblotting. b Phosphorylation of HSF1-S419 promotes cell survival. Cells, in which endogenous HSF1 was replaced with GFP, wild-type hHSF1-HA, or hHSF1-S419A mutants were heat-shocked at 45 °C for 4 h. Number of viable cells excluding trypan blue was counted, and MTT assay was performed. Extracts of cells were subjected to immunoblotting. c Phosphorylation of HSF1-S419 inhibits the accumulation of ubiquitylated proteins. Cells treated as described in b were heat-shocked at 45 °C for 4 h. The accumulation of insoluble ubiquitylated proteins was examined by immunoblotting using anti-Ub antibody and quantified. β-Actin levels in the soluble fraction were also shown. d TRRAP promotes refolding of the luciferase sensor protein during recovery from heat shock. HeLa-Fluc cells treated as described in a were heat-shocked at 42 °C for 2 h. These cells were then recovered at 37 °C for the indicated periods, and luciferase activity values were calculated and normalized to the value of control cells (100%). Extracts of cells were subjected to immunoblotting. e Phosphorylation of HSF1-S419 promotes refolding of the luciferase sensor protein. HeLa-Fluc cells treated as described in b were heat-shocked at 42 °C for 2 h. These cells were then recovered at 37 °C for 4 h, and luciferase activity values relative to that of control cells were estimated. Extracts of cells were subjected to immunoblotting. Norminal p values were determined by two-way ANOVA in a and d or by one-way ANOVA, followed by Tukey-Kramer test in b, c and e. Error bars indicate SEM (n = 4) in a and b, or (n = 3) in c, d, and e.