Stabilization of cytosolic Lyn complex by synergic cooperation of SH3 and catalytic domains. Cytosol from freshly isolated B-CLL cells lysed by sonication was subjected to the separation procedure described in Figure 2A and treated without or with 0.1 μM GA, 0.1 μM GST/SH3-Lyn, and 0.1 μM Pro-rich peptide, respectively, in the absence or presence of 10 μM lactacystin for 1 hour at 37°C. (A) Aliquots of each treated sample were analyzed for in vitro Lyn activity on Src-specific peptide substrate cdc2(6-20) and by Western blotting for Lyn. (B) Aliquots of each sample were immunoprecipitated with anti-Lyn antibody and the immunocomplexes probed with anti-HS1, anti–SHP-1/1L, and anti-Hsp90 antibodies, 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. The statistical analyses were performed using a one-way analysis of variance with posttest, and the significance is indicated as a P value. *P < .05, **P < .001, compared with control (bar 1). Data are representative of experiments performed in triplicate on samples from 16 B-CLL patients.