Protein phosphatase-1 (PP1) dephosphorylates PFN1 at Ser-137.

<p><i>A</i>, HEK293 cells were transfected with control, or sequence-specific siRNAs targeting human PP1Cα or PP2ACα. They were immunostained with P3490 (a–c) or the antibody against total PFN1 (d–f) (both in green) and counterstained with DAPI (blue). Silencing PP1Cα, but not PP2ACα, increased the number of P3490-positive cells and their staining intensities, while having no effect on total PFN1 levels. <i>B</i>, effects of PP1Cα and PP2ACα knockdown on P3490 staining were quantified using the fluorescence plate reader, normalized vs. DAPI, and represented as change relative to control siRNA. PP1Cα knockdown increased P3490 staining three-fold (***, p<0.001, unpaired t-test), while PP2ACα knockdown had no effect. Error bars represent the SEM. Data are mean ± S.E.M. of three independent experiments. <i>C</i>, Western blot confirmed >90% knockdown of both PP1Cα and PP2ACα in HEK293 cells. Neither affected PFN1 levels. Arrowhead indicates PP1Cα. The identity of the protein band above PP1Cα, which cross-reacts with PP1Cα antibody, is unknown. <i>D</i>, HeLa cells were transfected with Myc-PP1Cα(wt) (a–c), Myc-PP1Cα(H125A) (catalytically inactive) (d–f) or Myc-PP2ACα (g–i), double immunolabeled with an anti-Myc antibody (red) and P3490 (green), and counterstained with DAPI (blue). Neither Myc-PP1Cα(H125A) nor Myc-PP2ACα over-expression affected P3490 staining of the transfected cells (as indicated by the presence of cells that were both red and green). However, PP1Cα(wt) over-expression inhibited P3490 staining (as indicated by the mutual exclusivity of red and green cells).