Figure 4
From: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis
IER5 is a novel activator of HSF1.
(A–C) Control or HSF1-targeting siRNAs were introduced into H1299 cells. Subsequently, cells were transfected with control vector or an IER5 expression vector 24 hrs post-siRNA transfection. Cells were harvested 24 hrs post-DNA transfection. Expression of HSPA1A (A), HSPA6 (B) and HSF1 (C) mRNAs were analyzed by quantitative RT-PCR. (#p < 0.0001). (D) 293T cells were transfected with HA-HSF1 and control vector or IER5-Flag. Cells were harvested 24 hrs post-transfection. Cell lysates were crosslinked by DSP (1 mg/ml) and immunoprecipitated using anti-HA antibody. HSP90 binding to HA-HSF1 was detected by Western blotting. (E) H1299 cells were transfected with control vector or IER5-Flag expression vector. Cells were harvested 25 hrs post-transfection. Whole cell lysates were crosslinked with EGS. HSF1 and IER-Flag expression was analyzed by Western blotting. (F) H1299 cells were transfected with control vector, IER5-Flag, mut 1-Flag expression vector. Cells were harvested 24 hrs post-transfection. Subcellular localizations of endogenous HSF1 (detected using anti-HSF1 antibody), IER5-Flag and mut 1 (detected using anti-Flag antibody) were analyzed. HSF1 and IER5 expression levels were quantitated and shown at the bottom. (G) HSF1 binding to Heat Shock Elements (HSEs) was analyzed by streptavidin pull-down assay. Biotinylated control or HSE-containing DNA probes were bound to streptavidin-coated beads and mixed with control or IER5-expressing cell lysates. Bead-bound HSF1 was denatured in Laemmli buffer and analyzed by Western blotting.
