SH3 domain of cytosolic Lyn binds to HS1 and SHP-1L proteins. (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, anti-SHP-1/1L, anti-SHP-1, anti-HS1, anti-STAT3, anti-Akt, anti-Cbl, and anti-SHP-2 antibodies. Whole cell lysates from 2 × 105 B-CLL cells were probed with the same antibodies as positive controls. Downward arrows represent position of molecular weight standards on glycerol gradients, glutamate dehydrogenase (62 kDa), alcohol dehydrogenase (150 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa; Sigma-Aldrich), indicated to estimate the molecular weight of the protein complexes on parallel gradient runs. (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 GST/SH3-Lyn and 0.1 μM Pro-rich peptide, further subjected to immunoprecipitation by anti–SHP-1/1L and anti-Lyn antibodies, and assayed for Lyn and SHP-1/1L, respectively. (C) Same fractions as in panel B were collected, and aliquots were treated for 30 minutes at 4°C in the absence or presence of 0.1 μM GST/SH3-Lyn, 0.1 μM HS1ΔN-term, 0.1 μM HS1ΔSH3, 0.1 μM HS1-ΔPro-rich, 0.1 μM Pro-rich peptide, further subjected to immunoprecipitation by anti-HS1 and anti-Lyn antibodies, and assayed for Lyn and HS1, respectively.
