American Society of Hematology

Figure 5

$Figure 5. Exposure to CGP76030 triggers cell death of SUDHL6, Granta-519, DoHH2, VAL, Jeko-1, OCI-Ly3, and SR786 cell lines, blocks tyrosine phosphorylation of both PAG and Lyn in rafts, and dissociates Lyn from PAG and rafts. (A) Death of Raji, DoHH2, SUDHL6, Val, OCI-Ly3, Granta-519, Jeko-1, SR786, and L428 cells in presence of CGP76030 or PP2 at the indicated concentrations after 48 hours. These results are representative of 3 independent experiments. (B) Measurement of the proliferation rate of DoHH2 cells, loaded with CFSE, after CGP76030 exposure for 48, 72, and 96 hours. M1 corresponds to cells that have least proliferated, and M3 to cells with the highest proliferation rate. The results shown in panel B are representative of 3 independent experiments. (C-E) CGP76030 selectively dephosphorylates Lyn and PAG in rafts. (C) DoHH2 cells were cultured for 7 hours in the presence of 10 μM CGP76030, lysed in SDS sample buffer and WB with anti-P-Tyr antibody (lysate, left panel), or subjected to sucrose gradient fractionation in the presence of TX-100 as in Figure 1A. R and NR material containing equal amounts of Lyn was also probed with anti-P-Tyr antibody (right panel). (D) R and NR material (as in panel C) containing equal amounts of PAG were WB with anti-pY317 PAG, anti-PAG (MEM-255), and anti-pSTAT3 antibodies. (E) R and NR material (as in panel C) containing equal amounts of Lyn, was WB with anti-pY418 Src, anti-pY505 Lck, or anti-Lyn antibodies. Results in panels C through E are representative of 2 independent experiments. (F) CGP76030 redistributes Lyn of the raft fractions but minimally affects PAG localization. Equal volumes of sucrose gradient fractions of CGP76030-treated DoHH2 cells were WB with anti-PAG MEM-255 or anti-Lyn antibodies. Intensities were measured with ImageQuant and expressed as arbitrary units. Identical results were obtained in 2 independent experiments.$

Exposure to CGP76030 triggers cell death of SUDHL6, Granta-519, DoHH2, VAL, Jeko-1, OCI-Ly3, and SR786 cell lines, blocks tyrosine phosphorylation of both PAG and Lyn in rafts, and dissociates Lyn from PAG and rafts. (A) Death of Raji, DoHH2, SUDHL6, Val, OCI-Ly3, Granta-519, Jeko-1, SR786, and L428 cells in presence of CGP76030 or PP2 at the indicated concentrations after 48 hours. These results are representative of 3 independent experiments. (B) Measurement of the proliferation rate of DoHH2 cells, loaded with CFSE, after CGP76030 exposure for 48, 72, and 96 hours. M1 corresponds to cells that have least proliferated, and M3 to cells with the highest proliferation rate. The results shown in panel B are representative of 3 independent experiments. (C-E) CGP76030 selectively dephosphorylates Lyn and PAG in rafts. (C) DoHH2 cells were cultured for 7 hours in the presence of 10 μM CGP76030, lysed in SDS sample buffer and WB with anti-P-Tyr antibody (lysate, left panel), or subjected to sucrose gradient fractionation in the presence of TX-100 as in Figure 1A. R and NR material containing equal amounts of Lyn was also probed with anti-P-Tyr antibody (right panel). (D) R and NR material (as in panel C) containing equal amounts of PAG were WB with anti-pY317 PAG, anti-PAG (MEM-255), and anti-pSTAT3 antibodies. (E) R and NR material (as in panel C) containing equal amounts of Lyn, was WB with anti-pY418 Src, anti-pY505 Lck, or anti-Lyn antibodies. Results in panels C through E are representative of 2 independent experiments. (F) CGP76030 redistributes Lyn of the raft fractions but minimally affects PAG localization. Equal volumes of sucrose gradient fractions of CGP76030-treated DoHH2 cells were WB with anti-PAG MEM-255 or anti-Lyn antibodies. Intensities were measured with ImageQuant and expressed as arbitrary units. Identical results were obtained in 2 independent experiments.

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