Figure 1.

Hsp90 inhibition affects newly synthesized, but not mature, Src-kinases. (A—C) Cells were labeled with [³⁵S]methionine/cysteine for 3.5 h in the absence (−) or presence of 5 μM geldanamycin (GA) or of 10 μM radicicol (Rad). Cell lysates were analyzed by immunoprecipitation (ip; A), immunoblotting (B), or autoradiography (C). SupT1 cells were used for the detection of Lck, Daudi cells were used for Lyn, and transfected NIH-3T3 fibroblasts were used for Src. (A) Eighty percent of the volume of each lysate was used for immunoprecipitations. The anti-Lck serum recognizes in addition to Lck a nonrelated, slower-migrating protein (★) (Bijlmakers and Marsh, 1999). Lyn exists as two splice variants of 53 and 56 kDa (Yi et al., 1991). (B) Ten percent of the volume of each lysate was used for immunoblotting. (C) The blot shown in B for Daudi lysates was exposed to film to detect total labeled proteins. Molecular weight markers are indicated in kilodaltons on the right. (D) Lck was immunoprecipitated from unlabeled SupT1 cells treated with or without Hsp90 inhibitors as described above. Immunoprecipitates were analyzed for kinase activity. Autophosphorylated Lck is shown.