Figure 2
Figure 2. Purification and characterization of Lyn complex from cytosol of B-CLL. (A) Cytosol from 15 × 106 freshly isolated B-CLL cells lysed by sonication (top panel, S) or, alternatively, by douncing (bottom panel, D) was loaded on top of a linear glycerol gradient (10%-40%) and centrifuged 18 hours at 100 000g in an SW60Ti rotor (Beckman Coulter) at 4°C. Eighteen fractions (200 μL each) were collected from top and analyzed by immunoblotting with anti-Lyn antibody. The figure is representative of experiments performed in triplicate on samples from each of 5 B-CLL patients. (B) Cytosol, from 15 × 106 freshly isolated B-CLL cells lysed by sonication, was treated without (upper panel, −λPPase) or with λPPase (bottom panel, + λPPase), and subjected to the separation procedure described in panel A. Eighteen fractions (200 μL each) were collected from top, assayed for Lyn activity tested on Src-specific peptide substrate cdc2(6-20), and analyzed by immunoblotting with anti-pYA antibody and, after stripping, with anti-Lyn antibody. The figure is representative of experiments performed in triplicate on samples from each of 40 B-CLL patients. (C) Fractions 13 and 14 (CL complex) of the cytosol purified from 75 × 106 B-CCL cells and subjected to a linear glycerol gradient under the conditions described in panel A were collected and split into 5 aliquots, which were incubated for 30 minutes at 4°C in the absence (control) or presence of 0.05% SDS, 0.1 μM GST-Lyn/SH3, 0.1 μM GST-Lyn/SH2, and 0.1 μM GA, respectively. The treated samples were then subjected separately to glycerol gradient centrifugation as described in panel A, and aliquots of the resulting fractions were assayed for Lyn activity tested on Src-specific peptide substrate cdc2(6-20), and analyzed by immunoblotting, with anti-Lyn antibody. The figure is representative of experiments performed in triplicate on samples from 10 B-CLL patients. 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.

Purification and characterization of Lyn complex from cytosol of B-CLL. (A) Cytosol from 15 × 106 freshly isolated B-CLL cells lysed by sonication (top panel, S) or, alternatively, by douncing (bottom panel, D) was loaded on top of a linear glycerol gradient (10%-40%) and centrifuged 18 hours at 100 000g in an SW60Ti rotor (Beckman Coulter) at 4°C. Eighteen fractions (200 μL each) were collected from top and analyzed by immunoblotting with anti-Lyn antibody. The figure is representative of experiments performed in triplicate on samples from each of 5 B-CLL patients. (B) Cytosol, from 15 × 106 freshly isolated B-CLL cells lysed by sonication, was treated without (upper panel, −λPPase) or with λPPase (bottom panel, + λPPase), and subjected to the separation procedure described in panel A. Eighteen fractions (200 μL each) were collected from top, assayed for Lyn activity tested on Src-specific peptide substrate cdc2(6-20), and analyzed by immunoblotting with anti-pYA antibody and, after stripping, with anti-Lyn antibody. The figure is representative of experiments performed in triplicate on samples from each of 40 B-CLL patients. (C) Fractions 13 and 14 (CL complex) of the cytosol purified from 75 × 106 B-CCL cells and subjected to a linear glycerol gradient under the conditions described in panel A were collected and split into 5 aliquots, which were incubated for 30 minutes at 4°C in the absence (control) or presence of 0.05% SDS, 0.1 μM GST-Lyn/SH3, 0.1 μM GST-Lyn/SH2, and 0.1 μM GA, respectively. The treated samples were then subjected separately to glycerol gradient centrifugation as described in panel A, and aliquots of the resulting fractions were assayed for Lyn activity tested on Src-specific peptide substrate cdc2(6-20), and analyzed by immunoblotting, with anti-Lyn antibody. The figure is representative of experiments performed in triplicate on samples from 10 B-CLL patients. 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.

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