Evaluation of extracellular tyrosine phosphorylation of proteins in human platelets. (A) p-Tyr protein levels in total (Input) and p-Tyr immunoprecipitated fraction (p-Tyr IP) from platelet lysates were assessed by using western blot analysis. Phenyl phosphate (PP) was used as negative control for nonspecific binding to p-Tyr beads, and Ponceau S staining was used to examine total protein levels in input samples. Asterisks indicate activation-dependent tyrosine phosphorylation. (B) Phosphorylated tyrosine peptides isolated by anti–p-Tyr IP of tryptic digests from thrombin receptor activating peptide (TRAP)-stimulated human platelet lysates were analyzed by using liquid chromatography/tandem mass spectrometry. A total of 213 unique phosphopeptides were identified; within this pool, 4 tyrosine phosphosites were mapped to proteins with signal peptides or extracellular domains on transmembrane proteins as annotated in UniprotKB. (C) ENTPD6 (CD39L2) levels were determined in total (Input) and p-Tyr IP fraction in lysates from human platelets. Total protein levels in input were determined with Ponceau S staining. Tyrosine phosphorylation of V5 immunoprecipitates in RIPA-resistant extracts (SDS) from 293T cells coexpressing V5-tagged human (D) or mouse (E) ENTPD6 with wild-type (WT) or kinase-dead (KM) VLK. Extracts (Input) were analyzed for VLK and actin. Blue and black arrows indicate bands corresponding to ENTPD6 and VLK, respectively.