Phosphorylation of PKC substrates is dysregulated in VWD2B(p.V1316M) platelets. (A) Immunoblotting for phosphorylated PKC substrates following sodium dodecyl sulfate–polyacrylamide gel electrophoresis of platelet lysates from WT-VWF/WT and WT-VWF/p.V1316M mice (resting and PAR4p activated [200 μM]). β-actin was detected in every sample as loading control. (B) Quantification of PKC substrate phosphorylation (whole lane), normalized to β-actin content, using Image Studio Lite software (LI-COR Biosciences). Data are shown as arbitrary units (a.u.); PKC substrate phosphorylation in resting WT-VWF/WT platelets was set at 1. Data are mean ± SD, n = 5-8 mice per group. (C) Immunoblotting for phosphorylated PKC substrates following sodium dodecyl sulfate–polyacrylamide gel electrophoresis of human platelet lysates (resting) from 1 patient affected by VWD (p.V1316M) type 2B and a healthy donor (control). 14.3.3ζ (anti-14.3.3 ζ 4.; Santa Cruz Biotechnology) was detected as loading control. Horseradish peroxidase–conjugated secondary antibodies were applied, and immunoreactive bands were revealed using Pierce ECL Western Blotting Substrate. (D) Quantification of PKC substrate phosphorylation, normalized to loading control, performed with ImageJ software. Data are shown as a.u.; PKC substrate phosphorylation in resting control platelets was set at 1. Data are mean ± SD of 3 patients and 3 healthy controls. Immunoblots for patients 2 and 3 are reported in supplemental Figure 7. *P ≤ .05; ***P ≤ .001.