![Fig. 1. Detection of CPI, an inhibitory phosphoprotein of myosin phosphatase, in human platelets. / (A) Immunoblot analysis of CPI in human platelets. Platelet lysates were applied to SDS-PAGE, and this was followed by immunoblotting using an anti–CPI-17 antibody. Lane 1, smooth muscle CPI-17 purified from porcine aorta. Lane 2, platelet CPI. Molecular weight markers are indicated on the left end. (B) Co-immunoprecipitation of platelet myosin phosphatase with anti–CPI-17 antibody. Immunoprecipitates of platelet lysates with anti–CPI-17 antibody (lane 1) or with the preimmune serum (lane 2) were immunoblotted with anti–CPI-17 antibody. Immunoprecipitates with anti–CPI-17 antibody were immunoblotted using antibodies against the PP1δ catalytic subunit and MBS, respectively. (C) Immunoprecipitates with anti–CPI-17 antibody were assayed for phosphatase activity, using [γ-32P]-phosphorylated MLC as a substrate. Data represent the mean ± SE of 3 experiments. (D) In vitro platelet CPI phosphorylation by PKC. Anti–CPI-17 immnoprecipitates from platelet lysates were incubated for 2 minutes, without or with GF109203X, in the presence of purified platelet PKC. Protein phosphorylation was analyzed by SDS-PAGE, and this was followed by immunoblot analysis using an anti–pThr38–CPI-17 antibody. Results were expressed as fold increase in the phosphorylation relative to the activity measured in the absence of PKC. Data represent the mean ± SE of 3 experiments. (E) Effect of CPI thiophosphorylation by PKC on platelet myosin phosphatase activity. Platelet anti–CPI-17 immunoprecipitates were incubated, with or without purified platelet PKC, in the presence or absence of 5 μM GF109203X for 60 minutes at 30°C, as described in “Materials and methods.” Aliquots of the reaction mixture were quenched by adding a solution that resulted in a final concentration of 12 mM EGTA and 2 mM EDTA to stop the reaction, then were kept on ice. Myosin phosphatase activity was immediately determined. Data represent the mean ± SE of 4 experiments. IP, immunoprecipitation antibody used; IB, immunobotting antibody used; Ig, cross-reacting immunoglobulin.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/97/12/10.1182_blood.v97.12.3798/6/h81211168001.jpeg?Expires=1724237971&Signature=oAGGRMzdVKQe~FjChI6~gZ5Z15SEY18LTBJsXiXXJNIni0oXex1G54vTVOXdTtbzvrBxDE6~vuuNwjl~PILCQblZmbLgECgNtl7JwsjYH-FjYjcSPfS07y~b2EUEalOwQbHscU7XTXttKS2NE4K9PbKW1Mcyx56P1~sseJUIdSG75ut5Vy80utgyCceyvYtrL~T7C-rI00q1M-7IIWJxZwcdKq2CnA0ya95kq5XVEpPbRKqRHFLr9VOblXt3KiSFuPerwstice0ru9QjbT9AAaqB-KLp3~e9KM1s8niP1Rm1k-V4pYGXIfeOQuR95ni97IkBknejSS7VPHOHsXT2mg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Detection of CPI, an inhibitory phosphoprotein of myosin phosphatase, in human platelets.
(A) Immunoblot analysis of CPI in human platelets. Platelet lysates were applied to SDS-PAGE, and this was followed by immunoblotting using an anti–CPI-17 antibody. Lane 1, smooth muscle CPI-17 purified from porcine aorta. Lane 2, platelet CPI. Molecular weight markers are indicated on the left end. (B) Co-immunoprecipitation of platelet myosin phosphatase with anti–CPI-17 antibody. Immunoprecipitates of platelet lysates with anti–CPI-17 antibody (lane 1) or with the preimmune serum (lane 2) were immunoblotted with anti–CPI-17 antibody. Immunoprecipitates with anti–CPI-17 antibody were immunoblotted using antibodies against the PP1δ catalytic subunit and MBS, respectively. (C) Immunoprecipitates with anti–CPI-17 antibody were assayed for phosphatase activity, using [γ-32P]-phosphorylated MLC as a substrate. Data represent the mean ± SE of 3 experiments. (D) In vitro platelet CPI phosphorylation by PKC. Anti–CPI-17 immnoprecipitates from platelet lysates were incubated for 2 minutes, without or with GF109203X, in the presence of purified platelet PKC. Protein phosphorylation was analyzed by SDS-PAGE, and this was followed by immunoblot analysis using an anti–pThr38–CPI-17 antibody. Results were expressed as fold increase in the phosphorylation relative to the activity measured in the absence of PKC. Data represent the mean ± SE of 3 experiments. (E) Effect of CPI thiophosphorylation by PKC on platelet myosin phosphatase activity. Platelet anti–CPI-17 immunoprecipitates were incubated, with or without purified platelet PKC, in the presence or absence of 5 μM GF109203X for 60 minutes at 30°C, as described in “Materials and methods.” Aliquots of the reaction mixture were quenched by adding a solution that resulted in a final concentration of 12 mM EGTA and 2 mM EDTA to stop the reaction, then were kept on ice. Myosin phosphatase activity was immediately determined. Data represent the mean ± SE of 4 experiments. IP, immunoprecipitation antibody used; IB, immunobotting antibody used; Ig, cross-reacting immunoglobulin.
