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.

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.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal