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. 2017 Jun 15;102(8):1401–1412. doi: 10.3324/haematol.2016.155747

Figure 5.

Figure 5.

Effect of nintedanib on the phosphorylation state and activity of procaspase 8 and PP2Ac. (A) Western blotting (Wb) analysis with anti-pY380-procasp8 antibody, stripped and reprobed with anti-procasp8 antibody and with anti-β-actin antibody as a loading control, of total cell lysates of CLL cells from ten patients belonging to the various clinical and biological subtypes transfected by nucleofection with either scrambled siRNA (left-hand panels) or SHP-1-siRNA (right-hand panels) and cultured in the presence of increasing concentrations of nintedanib for 1 h. Densitometric analysis (arbitrary units) of the pY380-procasp8 and procasp8 bands is represented as histograms. (B) In vitro casp8 activity on cell lysates from CLL cells treated as in (A) as described in the Methods section. Compared with the effect of nintedanib, changes due to siRNA were statistically significant (*P≤0.01). (C) Wb analysis with anti-pY307-PP2Ac antibody, stripped and reprobed with anti-PP2Ac antibody and with anti-β-actin antibody as a loading control, of total cell lysates of CLL cells from ten patients treated as in (A). Densitometric analysis (arbitrary units) of the pY307-PP2Ac and PP2Ac bands is represented as histograms. Data are mean ± SD from four experiments performed in triplicate. (D) In vitro PP2A activity on cell lysates from CLL cells treated as in (A) by using a specific phosphopeptide as a substrate, as described in the Methods section of the Online Supplementary Data. Compared with the effect of nintedanib, changes due to siRNA were statistically significant (*P≤0.01).