Fig. 4.

PEST sequences interact with Cul3. a HSF2 interacts with Cul3 in yeast two-hybrid assay. β-galactosidase activity was measured in extracts of yeast carrying the pVP16-Cul3(161-293) plasmid and pGBD-HSF2 wild-type plasmid, pGBD-HSF2 in which one or both PEST domains were deleted, or empty pGBD-C1. Data are presented as means ± SEM (n = 3). b Extracts of cells transfected with GFP-HSF2 wild-type and GFP-HSF2 lacking the PEST domains were immunoprecipitated with anti-Cul3 or non-specific IgG antibodies followed by anti-GFP Western blot. c³⁵S-labeled in vitro translated Cul3 was incubated with GST-HSF2, GST-HSF2-P12 (GST fused to HSF2 sequence containing PEST domains 1 and 2), or GST bound to glutathione-agarose beads. After washing, amounts of bound ³⁵S-labeled Cul3 were determined by SDS-PAGE and autoradiography (upper panel). Amounts of GST-HSF2, GST-HSF2-P12, and GST bound to beads were measured by anti-GST Western blot (lower panel)