Figure 8

Western blot analysis of endogenous PPP1R2/PPP1R2P3 from human sperm. (A), Comparison of endogenous human sperm PPP1R2/PPP1R2P3, with recombinant PPP1R2 and PPP1R2P3, using sheep anti-PPP1R2 antibody. hsPPP1R2, heat-stable human sperm extract (both PPP1R2 and PPP1R2P3). Recombinant PPP1R2P3 and PPP1R2 are shown as positive controls. (B), Anti-PPP1R2 Western blot of 2D separation of hsPPP1R2 human sperm extract. Left panel, heat-stable human sperm extract; right panel, heat-stable human sperm extract supplemented with recombinant PPP1R2. Arrowheads indicate the three spots with different molecular mass and pI (C), hsPPP1R2 was incubated in the presence of different phosphatases, in the respective buffers at 30ºC for 3 hrs. 0, no phosphatase, control; PTP, incubation with Protein Tyrosine Phosphatase 1B; CIP, incubation with Calf Intestinal Phosphatase; PPP1, incubation with PPP1CC1. Recombinant PPP1R2 is shown as a positive control. (D), hsPPP1R2 was incubated in the presence of GSK3 (G) or CK2 (C), or both (G/C), as previously described. Resulting changes in protein migration, probably reflecting alterations in phosphorylation, were detected by Western blot analysis using the sheep anti-PPP1R2 antibody.