Figure 2

Uri is a PP1α specific binding protein. A. Uri binds all three Drosophila PP1α isozymes (PP1α13C, PP1α87B and PP1α96A) in yeast two-hybrid assays, but does not bind Drosophila PP1β (PP1β9C) in this assay. NIPP1 shows no discrimination between the isozymes, and is shown as a control. PP1 isoforms were expressed as DNA binding domain fusions (bait), while Uri and NIPP1 were Activation domain fusions (prey). B. Uri is an specific inhibitor of Drosophila PP1. The myelin basic protein phosphatase activity was measure in the presence of different concentrations of recombinant PP1α87B (triangles) or PP1β9C (squares). Phosphatase activity is shown as % of control in the absence of Uri. C. Uri immunoprecipitates ectopically expressed HA-PP1α87B more efficiently than HA-PP1β9C from fly extracts. Western blot showing similar levels of expression of HA-PP1α87B and HA-PP1β9C in total fly lysate, and proportion immuno-precipitated with the anti-Uri antibody. Normal guinea pig serum and Protein G sepharose only controls show no precipitation of the expressed proteins. D. Human PP1α (first lane) but not PP1β (second lane) co-immunoprecipitates with URI/RMP, and not URI/RMP-D2 (lanes 3 and 4) when co-expressed in COS7 cells (first panel). Expression controls are shown in the second and third panels.