Monitoring of Lyn-complex degradation during GA treatment. B-CLL cells were cultured in the presence of GA for different times, as described in Figure 6. (A) Cells were lysed by sonication in an isotonic buffer and subjected to differential centrifugation to separate microsomal and cytosolic fractions. Cytosol underwent glycerol gradient centrifugation, as described above. Fractions were collected from top and assayed for in vitro Lyn activity on Src-specific peptide substrate cdc2(6-20) and by Western blotting for Lyn. (B) Cytosol, isolated from B-CLL cells as in panel A, was immunoprecipitated with anti-Lyn antibody. Immunocomplexes were then probed with anti-HS1, anti–SHP-1/1L, and anti-Hsp90, respectively. Blots were then stripped and reprobed with anti-Lyn antibody. The bar graph above the blot panels represents the values of a densitometric analysis (arbitrary units) of anti-HS1, anti–SHP-1/1L, and anti-Hsp90 bands, expressed as mean plus or minus SD. Data are representative of 3 experiments performed with 8 B-CLL samples. (C) Proposed model for sequential binding of ligands in the assembly of the CL complex. Step 1 indicates SH3 binding proteins (X) can promote displacement of the PPII motif in the SH2-kinase linker from the SH3 domain, thus inducing an “open” conformation; Step 2, association of Hsp90 with the N-terminal lobe of Lyn catalytic domain stabilizes the complex and maintains the kinase in an active conformation.