PKD is expressed in human platelets and is activated by PMA, convulxin, thrombin, and U46619.
Washed platelets were treated with indomethacin (10 μM) and apyrase (2 U/mL), with the exception of those activated by U46619, which were treated with apyrase alone. (A) Whole cell lysates from platelets (12.5 × 106 cells) and Jurkat T cells (12.5 × 105 cells) were subjected to SDS-PAGE, transferred to PVDF membrane, immunoblotted with PA-1 antiserum, and visualized by chemiluminescence. Similar results were obtained using 2 other anti-PKD antibodies, sc639 and sc937 (data not shown). One experiment representative of 3 is shown. (B) Time courses of PKD activation were obtained using PMA (30 nM), convulxin (12 nM), thrombin (0.1 U/mL), and U46619 (3 μM). (i) Platelets were lysed and immunoprecipitated with the sc937 antiserum and PKD activity was determined by an in vitro kinase assay using peptide syntide-2 as substrate. Results are shown as the mean ± SEM and are representative of 3 independent experiments, each in duplicate. (ii) Lysates were analyzed by SDS-PAGE and Western blotting with the pS916 antibody, and the Western blots were reprobed with a pan–C-terminal PKD antibody (pan sc639). One experiment representative of 3 independent experiments is shown.
