American Society of Hematology

Figure 3

$Figure 3. Lyn-dependent tyrosine phosphorylation inhibits casp8 activity in B-CLL cells. (A) Whole B-cell lysates from 6 normal donors and all B-CLL patients distributed between the UM-CLL and M-CLL subsets (supplemental Table 1) were analyzed by Wb analysis with anti-procasp8 antibody, and the blots were reprobed with anti-β-actin antibody as a loading control. Pooled densitometric analysis (arbitrary units) of the Wb bands of the immunoblots are shown (left panel). Wb analysis representative of one normal donor and B-CLL patients equally distributed between the UM-CLL (patients 9, 17, and 32) and M-CLL subsets (patients 2, 21, and 38) are shown in the right panel. (B) Equivalent amounts of protein of the different subcellular fractions (first particulate fraction, I-Par., corresponding to nuclei and mitochondria; second particulate fraction, II-Par., corresponding to microsomes; and cytosol) from normal donors and B-CLL patients belonging to the UM-CLL and M-CLL subsets (represented by patients 5 and 13, respectively, compared with one normal donor in this figure) were analyzed by Wb analysis with anti-procasp8 antibody, and the blots were reprobed with antibodies against lactate dehydrogenase (LDH, cytosolic marker), plasma membrane Ca2+ ATPase (PMCA, plasma membrane marker), and lamin (nuclear marker) as loading controls. The figure is representative of samples from 3 normal donors and 10 CLL patients belonging to 2 different subsets UM-CLL and M-CLL composed of 5 samples each. (C-D) Cytosol from normal donors and B-CLL patients belonging to the UM-CLL and M-CLL subsets (patients 5 and 13, respectively, compared with one normal donor in this figure) were incubated in the absence (lanes 1, 3, and 5) or presence of λ-PPase (lanes 2, 4, and 6) for 1 hour at 37°C. Equivalent amounts of protein underwent Wb analysis with anti-p-procasp8 and anti-procasp8 antibodies (C) or assayed for in vitro casp8 activity (D). The figure is representative of samples from 3 normal donors and 20 B-CLL patients belonging to the UM-CLL and M-CLL subsets composed of 10 samples each (*P < .05, lanes 3 vs 4 and 5 vs 6).$

Lyn-dependent tyrosine phosphorylation inhibits casp8 activity in B-CLL cells. (A) Whole B-cell lysates from 6 normal donors and all B-CLL patients distributed between the UM-CLL and M-CLL subsets (supplemental Table 1) were analyzed by Wb analysis with anti-procasp8 antibody, and the blots were reprobed with anti-β-actin antibody as a loading control. Pooled densitometric analysis (arbitrary units) of the Wb bands of the immunoblots are shown (left panel). Wb analysis representative of one normal donor and B-CLL patients equally distributed between the UM-CLL (patients 9, 17, and 32) and M-CLL subsets (patients 2, 21, and 38) are shown in the right panel. (B) Equivalent amounts of protein of the different subcellular fractions (first particulate fraction, I-Par., corresponding to nuclei and mitochondria; second particulate fraction, II-Par., corresponding to microsomes; and cytosol) from normal donors and B-CLL patients belonging to the UM-CLL and M-CLL subsets (represented by patients 5 and 13, respectively, compared with one normal donor in this figure) were analyzed by Wb analysis with anti-procasp8 antibody, and the blots were reprobed with antibodies against lactate dehydrogenase (LDH, cytosolic marker), plasma membrane Ca2⁺ ATPase (PMCA, plasma membrane marker), and lamin (nuclear marker) as loading controls. The figure is representative of samples from 3 normal donors and 10 CLL patients belonging to 2 different subsets UM-CLL and M-CLL composed of 5 samples each. (C-D) Cytosol from normal donors and B-CLL patients belonging to the UM-CLL and M-CLL subsets (patients 5 and 13, respectively, compared with one normal donor in this figure) were incubated in the absence (lanes 1, 3, and 5) or presence of λ-PPase (lanes 2, 4, and 6) for 1 hour at 37°C. Equivalent amounts of protein underwent Wb analysis with anti-p-procasp8 and anti-procasp8 antibodies (C) or assayed for in vitro casp8 activity (D). The figure is representative of samples from 3 normal donors and 20 B-CLL patients belonging to the UM-CLL and M-CLL subsets composed of 10 samples each (*P < .05, lanes 3 vs 4 and 5 vs 6).

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