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. 2011 Apr 28;117(22):5941–5952. doi: 10.1182/blood-2010-08-300772

Figure 4.

Figure 4

Regulation of Bcr-Abl1 expression levels by SK-1/S1P signaling via the modulation of PP2A. (A) Effects of the down-regulation of SK-1 using siRNA on Bcr-Abl1 protein in the absence/presence of okadaic acid (OA; 10nM) or SS (10μM), inhibitors of PP2A or SHP1, respectively, for 24 hours were determined by Western blotting, and the Scr-siRNA transfected samples were used as controls. Actin was used as a loading control. (B) Activation of PP2A in response to knockdown of SK-1 using siRNA in K562/IMA-3 cell extracts was determined ex vivo using the PP2A activity assay kit. (C) Roles of PP2A activation in the regulation of Bcr-Abl1 and P-Bcr-Abl1 in response to Scr or SK-1 siRNAs were examined in vector controls vs I2PP2A-GFP over-expressing K562/IMA-3 cells using Western blotting (first and second panels lanes 1-2 and 3-4, respectively). Effects of vector-only and I2PP2A overexpression on total and P-PP2A at Y307 (inactive form) in Scr or SK-1 siRNA treated K562/IMA-3 cells were also determined using Western blotting (third and fourth panels lanes 1-2 and 3-4, respectively). Actin was used as a loading control (fifth panel). (D) Role of I2PP2A overexpression in the regulation of cell viability in response to SK-1 siRNA was examined using trypan blue exclusion in drug resistant LAMA-4/IMA cells. (E) Effects of SK-1-V5 overexpression compared with vector-transfections (lanes 2 and 1, respectively) on the total PP2A or SHP1 vs inactive P-PP2A at Y307, or P-SHP1 at S591 (top left and right first and second panels, respectively) were measured by Western blotting. Effects of overexpression of SK-1 compared with vector transfections on the levels of P-Bcr-Abl1 at Y245 were determined by Western blotting (bottom left third panel lanes 2 and 1, respectively). Expression of SK-1-V5 was confirmed using the anti-V5 antibody in Western blotting (bottom right panel), and β-actin was used as a loading control (bottom left third panel). These experiments were performed in duplicates, and error bars represent SD. *P < .05 was considered significant. In panels C and E, relative expression levels of proteins (normalized to actin levels) are shown below each lane.