Figure 7. TRiC directly interacts with HSF1.

(A) HEK293T cells transfected with a plasmid expressing FLAG-HSF1 or an empty vector (Vec), were treated with DSP (+) or left untreated (−). HSF1 was immunoprecipitated from total protein extract using anti-FLAG-affinity resin and analyzed for HSF1, Hsp90, Hsp70, Cct2 and Cct3 by immunobloting. * indicates non-specific band. (B) HEK293T cells transfected with mouse HSF1-GFP-TAP or an empty vector were treated with DSP and HSF1 from total protein extract using anti-GFP-affinity resin. The immunoprecipitate was analyzed for HSF1, Hsp90, Hsp70, Cct3 by immunoblotting. (I, input; PD, pull-down) (C) Purified bovine TRiC, His₆-tagged HSF1ΔLZ1-3 or His₆-tagged wild-type HSF1 were incubated either alone or with the indicated combinations. Proteins were captured using cobalt-agarose resin and analyzed for HSF1, Cct2 and Cct3 by immunoblotting. Shown are images from the same blot that was separately probed with the indicated antibodies (D) In vitro TRiC – HSF1 interaction experiment carried out as in (C) but purified TRiC was pre-incubated with 200 μM HSF1A-Biotin or Biotin alone prior to addition of HSF1. (E) NIH3T3 and HEK293T cells transfected with a plasmid expressing FLAG-HSF1 were treated with 80 and 100 μM HSF1A, respectively, or DMSO for 6 h, cross-linked with DSP, HSF1 immunoprecipitated and immunoblotted as in (A) for HSF1, Hsp90, Hsp70 and Cct3. (F) Model for the repressive interaction between TRiC and HSF1 and its modulation by HSF1A.