Interaction of PTPN22 with p-PDE5A in activated platelets. Coimmunoprecipitation analysis of the relationship of PTPN22 with p-PDE5A or Csk (A) or the relationship of p-PDE5A with PTPN22 (B) in platelets after stimulation with CRP (5 μg/mL) for 5, 15, 30, or 60 seconds. (Similar results were obtained from 3 independent experiments). (C) Lysates of CRP-treated PTPN22-deficient platelets were incubated with GST-PTPN22 PTPase (0.4 μg) (in the presence or absence of 40 μM LTV-1), GST-PTPN22 C227S, or GST followed by measurement of PDE5A phosphorylation (Ser92) by western blot. —: indicates the PDE5A phosphorylation in CRP-treated platelets. (D) After CRP stimulation, PTPN22-deficient platelet lysates were incubated with GST or GST-PTPN22 PTPase followed by detection of the phosphorylation of PDE5A (Ser92) or Syk (Tyr519/520) by western blot. The phosphorylation of PDE5A or Syk after GST-PTPN22 PTPase treatment was quantified as a ratio of their phosphorylation level relative to GST (mean ± SD, n = 3 independent experiments). **P < .01. Phosphopeptide (1 μM) was incubated with increasing amounts of GST or GST-PTPN22 PTPase (0-16 ng) (E), or GST and GST-PTPN22 PTPase (4 ng) was incubated with the increasing amounts of peptide (0-4 μM) for 5 minutes at 37°C followed by the addition of 50 μL Malachite Green Reagent and measurement of the absorption value (mean ± standard error, n = 3 independent experiments) (F). (G) GST-PTPN22 PTPase (4 ng) was incubated with peptide (1 μM) in the absence (vehicle) or presence of LTV-1 (20 μM) or NC1 (20 μM) for 5 minutes followed by measurement of phosphate release. The phosphate concentration was calculated by using the phosphate standard and corrected by subtracting the values of GST protein. Mutant: PTPN22 C227S. Compared with vehicle, ***P < .001 (mean ± standard error, n = 3 independent experiments).