Figure 1: SSBP1 interacts with the trimerization domain of HSF1.

(a) Interaction between HSF1 and SSBP1. Total cell lysates and the nuclear and cytosolic fractions were prepared from MEF cells at 37 °C (HS-) and cells treated with heat shock at 42 °C for 60 min (HS+). Complexes co-immunoprecipitated using preimmune serum (Pl) or antiserum for HSF1 were blotted with SSBP1 or HSF1 antibody. Total proteins in each lysate were blotted using the same antibodies as a control (Input). They were also blotted using an antibody for a nuclear protein SP1 or HSP90 localized dominantly in the cytoplasm, and are shown at the bottom. (b) Trimerization domain of HSF1 is required for the interaction with SSBP1. GST pull-down assay from mixtures of each purified GST-fused hHSF1 protein with mSSBP1-His protein was performed, and blotted with SSBP1 or GST antibody (lower). Degraded recombinant proteins were observed in some lanes. Schematic representation of hHSF1 deletion mutants fused to GST is shown (upper). The ability of each protein to interact with SSBP1 is indicated on the right. DBD, DNA-binding domain; HR, hydrophobic heptad repeat; DHR, downstream of HR-C. A red bar indicates the trimerization domain (HR-A/B, amino acids 130–203), which is required for interaction with SSBP1. (c) Alignment of the amino-acid sequences of the trimerization domain of human HSF members. Residues identical among the three sequences are indicated in black, and those identical between two are in grey. Open and solid squares show the heptad repeats of hydrophobic amino acids. Seven residues indicated by dots were mutated (see Supplementary Fig. 1b). Among them, lysine at amino-acid 188 (K188; red dot) was required for the interaction with SSBP1. (d) Interaction of SSBP1 with HSF1 point mutants. Each Flag-tagged hHSF1 point mutant at K188 was expressed in HEK293 cells, and complexes co-immunoprecipitated using SSBP1 antibody were blotted with Flag or SSBP1 antibody. (e) DNA-binding activity of HSF1 point mutants. Whole-cell extracts were prepared from cells treated as described in d, and were subjected to EMSA using ³²P-labelled HSE-oligonucleotide in the presence of preimmune serum (Pl) or HSF1 antibody (Anti-HSF1) (upper). Western blotting was also performed (lower).