American Society of Hematology

Figure 5

$Figure 5. Effect of FTY720 on the association of the SET/PP2A complex. (A) Chemical structure of FTY720. (B) Freshly isolated CLL cells were cultured in the absence or presence of 50 µM pervanadate, supplemented with increasing concentration of FTY720 for 8 hours. After such treatment, each sample was immunoprecipitated with anti-PP2Ac antibody and the immunocomplexes were analyzed by western blot (Wb) analysis with anti-SET. Blots were then stripped and reprobed with anti-PP2Ac antibody. Pooled densitometric analyses (arbitrary units) of the Wb bands of the immunoblots are shown. (C) The interaction of SET and PP2A in pervanadate-treated and untreated CLL cells was measured as a ratio of the densitometric values of SET over those of PP2A as a function of FTY720 concentration. (D) Cytosol purified by freshly isolated CLL cells treated as previously described was loaded on top of a linear glycerol gradient (10%-40%) and centrifuged for 18 hours at 100 000g in an SW60Ti rotor (Beckman Coulter) at 4°C. Eighteen fractions (200 μL each) were collected from top and analyzed by immunoblotting with anti-PP2Ac and anti-SET antibody. The figure is representative of experiments performed in triplicate on samples from each of 16 CLL patients. Downward arrows represent the position of molecular weight standards on glycerol gradients, glutamate dehydrogenase (62 kDa), alcohol dehydrogenase (150 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa; Sigma-Aldrich), indicated to estimate the molecular weight of the protein. The figure is representative of experiments performed in triplicate on samples from each of 16 CLL patients.$

Effect of FTY720 on the association of the SET/PP2A complex. (A) Chemical structure of FTY720. (B) Freshly isolated CLL cells were cultured in the absence or presence of 50 µM pervanadate, supplemented with increasing concentration of FTY720 for 8 hours. After such treatment, each sample was immunoprecipitated with anti-PP2Ac antibody and the immunocomplexes were analyzed by western blot (Wb) analysis with anti-SET. Blots were then stripped and reprobed with anti-PP2Ac antibody. Pooled densitometric analyses (arbitrary units) of the Wb bands of the immunoblots are shown. (C) The interaction of SET and PP2A in pervanadate-treated and untreated CLL cells was measured as a ratio of the densitometric values of SET over those of PP2A as a function of FTY720 concentration. (D) Cytosol purified by freshly isolated CLL cells treated as previously described was loaded on top of a linear glycerol gradient (10%-40%) and centrifuged for 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-PP2Ac and anti-SET antibody. The figure is representative of experiments performed in triplicate on samples from each of 16 CLL patients. Downward arrows represent the 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. The figure is representative of experiments performed in triplicate on samples from each of 16 CLL patients.

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