Figure 2.

Effect of nintedanib on the activity of the differently phosphorylated forms of SHP-1 inside chronic lymphocytic leukemia cells. (A) Phosphatase activity of SHP-1 immunoprecipitated from the cytosol (left) and particulate (right) of CLL cells cultured in the absence or presence of increasing concentrations of nintedanib for 1 h measured as [³²P] released from [³²P]-Band 3. Data are mean ± SD of three experiments performed in triplicate. (B) Expression and phosphorylation state of SHP-1 of CLL cells cultured in the presence of 15 μM nintedanib over time. (C) Phosphatase activity of SHP-1 immunoprecipitated from the cytosol (left) or particulate (right) of the CLL cells described in (B) measured as [³²P] released from [³²P]-Band 3. (D) Densitometric analysis of western blots probed with anti-pS591 or anti-pY536 antibody (arbitrary units, open circles, left- and right-hand panel, respectively) and phosphatase activity from (C) normalized as percentage (solid circles, left and right panel, respectively) and plotted as line graphs. Data are mean ± SD of three experiments performed in triplicate (*P≤ 0.01), n=16. Wb: western blot; IP: immunoprecipitation; incub: incubation.