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. 2004 Jun 7;101(24):8888–8893. doi: 10.1073/pnas.0307812101

Fig. 4.

Fig. 4.

P-CPI-17 specifically inhibits myosin phosphatase. Phosphatase assays were carried out by using purified myosin phosphatase (A) or glycogen-bound PP1 (B), in the presence of unphosphorylated CPI-17 (open circle)-, phosphorylated CPI-17 (filled circle)-, or thiophosphorylated (filled triangle)-CPI-17 at the indicated concentrations. Phosphatase activity without added CPI-17 is set as 100%. Assay was initiated by addition of phosphatase preparation. Okadaic acid (1 nM) and EDTA (0.1 mM) were added in the reaction mixture to inhibit any PP2A and PP2B activities that possibly contaminated the glycogen-bound PP1 preparation. (C) Myosin phosphatase (M), glycogen-bound PP1 (G), and recombinant PP1C α isoform (α) were subjected to immunoblotting by using PP1C δ isoform specific antibody (Upper) and pan-PP1C antibody (Lower). (D) Aliquots of the inhibition assay mixture including phospho-CPI-17 (100 nM) were subjected to immunoblotting by using anti-P-CPI-17(T38) and anti-CPI-17 antibodies. Mean values from duplicate assays are shown. n indicates mixture without added phosphatases.