Fig. 5. Hsp90 function is required to maintain mature Hck499F in a catalytically active conformation. A, 293T cells expressing Hck499F were treated 48 h posttransfection with 100 µg/ml cycloheximide for 2 h and then with either DMSO or 2.5 µM GA for 0, 1, or 4 h. The cells were lysed and the whole cell lysates Western blotted with anti-phosphotyrosine (-pY) and anti-Hck antibodies. The positions of molecular mass markers (in kDa) are shown on the right. B, Hck499F was immunoprecipitated from aliquots of the whole cell lysates shown in A and subjected to an in vitro autophosphorylation reaction or Western blotting with an anti-Hck antibody. C, Hck499F was synthesized for 35 min at 30°C in rabbit reticulocyte lysates containing [35S]-methionine. Reinitiation of protein synthesis was inhibited, and reactions were incubated at 30°C for 60 min. Subsequently, DMSO (-) or GA (+) was added, and the reactions were incubated for an additional 60 min at 37°C. Then Hck499F was immunoprecipitated from aliquots of the rabbit reticulocyte lysates and subjected to an in vitro autophosphorylation reaction or directly subjected to autoradiography after SDS-PAGE. D, aliquots of the rabbit reticulocyte lysates from C were incubated with the indicated concentration of chymotrypsin, then subjected to SDS-PAGE and then autoradiography. The position of full-length [35S]-methionine-labeled Hck499F is indicated on the left.