FIG. 3.

Hsf4b is phosphorylated on serine and threonine residues. (A) H1299 cell lysates expressing empty vector or Flag-Hsf4b and different amounts of plasmids encoding HA-DUSP26 were analyzed by immunoblotting using antibody to Flag-Hsf4b, HA-DUSP26, or β-actin. (B) Hsf4b is a threonine-phosphorylated protein. H1299 cells were transiently transfected with Flag-Hsf4b before treatment with 0 to 100 μM of sodium vanadate for 2 h. Flag-Hsf4b was immunoprecipitated (500 μg of lysate) using anti-Flag antibody and visualized with antibodies to Flag-Hsf4b, phosphothreonine, or phosphotyrosine as indicated. β-Actin is shown to indicate that an equal amount of cell lysate was used for immunoprecipitation. (C) H1299 cells were transiently transfected with plasmids containing Flag-Hsf4b, with or without cotransfection with plasmids containing HA-DUSP26. Following 3 h of metabolic labeling with ³²P at 37°C, ³²P-Flag-Hsf4b was immunoprecipitated from equal amounts of cell lysate using antibody to Flag, and immunoprecipitated material was prepared for phosphoamino acid analysis in either the absence (left panel) or presence (right panel) of coexpressed HA-DUSP26. The positions of the phosphorylated threonine, serine, or tyrosine are indicated by arrowheads. Lower panel: immunoblotting experiments showing expression of Flag-Hsf4b (lanes 1 and 2) and HA-DUSP26 in the cell lysate. Hsf4b usually appears to be lower in quantity (dephosphorylated) when coexpressed with DUSP26 (lane 2). β-Actin is shown to indicate an equal amount of cell lysate in each lane. (D) DUSP26 affects Hsf4b binding to HSE. H1299 cells were cotransfected with Flag-Hsf4b and increasing amounts of HA-DUSP26 (same samples as in panel A, lanes 1 to 4). After 48 h, 10 μg of cell lysate was analyzed by EMSA. Lane 5 is the same cell lysate as in lane 1 plus antibody to Flag to show supershift of Hsf4b. Expression of proteins expressed in the cell lysates are as in panel A.