Figure 4.
Mapping the Hsp70 binding site within the HSF1 activation domain. (A) Schematic diagram of wild-type and deletion mutants of HSF1 activation domain. Constructs I (wild type), and II–V (deletion mutants) are indicated as the regions of HSF1 activation domain fused to GST. The boundaries of each construct and the levels of Hsp70-binding activity are indicated. (B) A potential Hsp70 binding site is located from amino acid residue 425 to 439 of the HSF1 activation domain. HSF1–GST fusion proteins were incubated with HeLa whole cell extracts (WCE). The presence of Hsp70 as bound protein was detected by Western blot analysis with Hsp70-specific antibody 3A3. Purified recombinant Hsp70 and an aliquot of HeLa whole cell extracts were included as positive controls. (C) SDS-PAGE of each purified GST fusion protein visualized by Coomassie blue staining. Molecular weight markers (MW) are included.