Phosphorylation state and protein level of the catalytic subunit of PP2A in CLL cells. (A) Whole B-cell lysates from 6 normal donors and from all CLL patients were analyzed by western blot (Wb) analysis with anti-pY307-PP2Ac antibody, and the blots were reprobed with anti-PP2Ac antibody as well as anti-β-actin antibody as a loading control (left panel). Wb analysis representative of 1 normal donor and CLL patients (patients 3, 12, 24, 29, 33, and 39) are shown in the right panel. (B) Freshly isolated CLL cells from 14 patients were cultured in the absence or presence of 0.1 µM dasatinib, 10 µM PP2, 1 µM saracatinib, or 10 nM GA at different time points. After such treatment, cells were either (1) lysed and analyzed by Wb analysis with anti-pY307-PP2Ac antibody and the blots were reprobed with anti-PP2Ac antibody (left panels) or (2) subject to a phosphatase activity assay by using pAkt, obtained by immunoprecipitation from CLL cells, for 10 minutes at 37°C. In the latter case, the samples were analyzed by Wb analysis with anti-p-Akt and anti-Akt antibodies. Pooled densitometric analyses (arbitrary units) of the Wb bands of the immunoblots are shown (right panels). Compared with the control, changes were statistically significant (*P < .01). (C) Freshly isolated CLL cells were transfected by nucleofection with either the negative control or Lyn-siRNAs and cultured for 48 hours in complete medium. Cell lysates underwent Wb analysis with anti-Lyn antibody (lanes 1and 2) and anti-pY307-PP2A (lanes 3 and 4) and were reprobed, after stripping, with anti-β-actin and anti-PP2Ac antibodies, respectively, as loading controls. Pooled densitometric analyses (arbitrary units) of the Wb bands of the immunoblots are shown (right panels). Compared with the control, changes were statistically significant (*P < .01) to assess the efficacy of siRNA technology.