Validation of the transactivation assay used, transactivation analysis of mutant S326A, and isolation of FLAG-HSF1 for phosphorylation analyses. (A) Transactivation assay of cells in 96-well dishes co-transfected with reporter gene mixture (LEXA-fLUC and pRL-CMV) and 0–40 ng LEXA-HSF1. C: Control unheated cells; HS: Cells subjected to standard heat-treatment for 30 min at 44°C. (B) Relative amounts of HSF1-immunoreactive protein in cells transfected with 0.5 ng β-galactosidase expression construct (B-GAL) or 0.5 ng LEXA-HSF1. Extracts prepared one day after transfection were analyzed by anti-HSF1 western blot. A quantitation of the HSF1 signals, shown in the inserts on top, is presented. Note that the insert only shows the relevant (scanned) portion of the blot, i.e., polypeptide sizes from about 60–100 kD. HSF1 forms appear as several closely spaced bands above a single band representing a nonspecific signal. (C) Coomassie Blue-stained gel containing immune-isolated FLAG-HSF1. M: markers having molecular weights in kD as indicated on the left. Note that the cluster of bands appearing at and below the 98 kD marker was identified as HSF1 forms by a parallel anti-HSF1 western blot (not shown). (D) Transactivation assay comparing activities of HSF1 forms in cells co-transfected with luciferase reporter gene mixture and expression constructs (0.5 ng) for FLAG-LEXA-HSF1 substitution mutant S326A, parent FLAG-LEXA-HSF1 or B-GAL. One day after transfection, cells were exposed either to 300 μM CdCl2 for 2 h (Cd) or to standard heat treatment (HS), or were left untreated (C). Relative firefly luciferase activities assayed 6 h after treatments are shown. (E) FLAG western blot of parallel (heat-treated) cultures from the experiment analyzed under C that compares expression levels of FLAG-LEXA-HSF1 mutant S326A and parent FLAG-LEXA-HSF1 one day after transfection. The tubulin signal was used as loading control. The numbers below the blots represent a quantitative comparison of the mutant S326A and parent FLAG-LEXA-HSF1 signals in the FLAG blot.
Guettouche et al. BMC Biochemistry 2005 6:4 doi:10.1186/1471-2091-6-4