Hsp90 is detectable in Lyn complex and interacts with Lyn. (A) Cytosol from 15 × 106 freshly isolated B-CLL cells lysed by sonication was subjected to the separation procedure described in Figure 2A. Fractions 13 and 14 (CL complex) were collected and resubmitted to an additional centrifugation step on a glycerol gradient. Aliquots of the gradient fractions were analyzed by immunoblotting with anti-Lyn and anti-Hsp90 antibodies. Downward arrows represent position of molecular weight markers on glycerol gradient. (B) CL complex purified after 2 centrifugation steps on a glycerol gradient, as described in panel A, was collected, and aliquots were treated for 30 minutes at 4°C in the absence or presence of 0.1 μM GA, 0.1 μM 17-AAG, 0.1 μM GST/SH3-Lyn, and 0.1 μM Pro-rich peptide, respectively, and further subjected to immunoprecipitation by anti-Hsp90 or anti-Lyn antibodies. Immunoprecipitates were subsequently assayed both for Lyn and Hsp90, respectively. The figure is representative of experiments performed in triplicate on samples from 40 B-CLL patients.
