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. 2003 Apr;23(8):2953–2968. doi: 10.1128/MCB.23.8.2953-2968.2003

FIG. 6.

FIG. 6.

Phosphorylation of S303/7 occurs concomitantly with sumoylation of HSF1. (A) K562 cells were cotransfected with Myc-tagged wild-type HSF1 and GFP-SUMO-1 and heat shocked for the indicated time periods. Cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-HSF1 antibody to determine the level of HSF1 sumoylation. Equal loading was confirmed by anti-Hsp90 immunoblotting. (B) HSF1 was immunoprecipitated from the same samples as for panel A with a monoclonal anti-HSF1 antibody, and the immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-p-S303/7-HSF1 and anti-HSF1 antibodies. (C) Myc-tagged wild-type HSF1, S303/7A, or S303/7D was cotransfected with GFP-SUMO-1 into K562 cells followed by a 15-min heat shock at 42°C. Cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-HSF1 antibody. Equal loading was confirmed by anti-Hsp90 immunoblotting. WT, wild type; WB, Western blot; IP, immunoprecipitation. Numbers on right of panels A and C are molecular masses in kilodaltons.