Fig. 4. Hsp90 function is required for the synthesis of catalytically active Hck499F. A, 293T cells were transfected with empty vector (-) or pEF-Hck499F (+) for 4 h, then immediately treated with DMSO (-) or 2.5 µM GA (+) for 16 h. The cells were lysed, and the whole cell lysates (WCL) were 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 and supplemented with either DMSO (-) or GA (+). Hck499F was then 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.