Figure 4: Nuclear translocation of SSBP1 is dependent on HSF1.

(a) Nuclear translocation of SSBP1 is inhibited by HSF1 knockdown. HeLa cells were infected with Ad-sh-SCR or Ad-sh-hHSF1-KD1 for 72 h, and then treated without (Cont.) or with heat shock at 42 °C for 60 min (HS). The cells were co-stained with antibodies for SSBP1 (green), TOM20 (red) and DAPI (blue), and fluorescence images were merged (Merge; upper). Scale bars, 10 μm. Cell extracts in NP-40 lysis buffer were prepared and subjected to western blotting using an antibody for HSF1 or β-actin (lower). (b) Subcellular fractionation. HeLa cells were treated as described in a. Cytoplasmic (C), nuclear (N) and mitochondrial (M) fractions were prepared as described in Methods. Equivalent volumes of the fractions were subjected to western blotting using antibodies for SSBP1, HSP90, SP1 and TOM20. (c) Subcellular localization of SSBP1 in cells overexpressing HSF1 or its mutant. Cells were infected with adenovirus expressing Ad-sh-hHSF1-KD1 or Ad-sh-SCR for 24 h, and were then infected for 48 h with adenovirus expressing wild-type HSF1 (WT), each point mutant or LacZ. These cells were heat-shocked at 42 °C for 60 min, and co-stained with antibodies for SSBP1 (green) and Flag tag (red), and with DAPI (blue), and fluorescence images were merged (Merge; upper). Cell extracts were prepared and subjected to western blotting using antibody for HSF1 or β-actin (lower). Scale bars, 10 μm. (d) Subcellular fractionation was performed using the cells treated as described in c, and fractions of the cytoplasm (C), nucleus (N) and mitochondria (M) were subjected to western blotting as described in b.