Subcellular localization of Nbeal2 and its interactors, Dock7, Sec16a, and Vac14 in platelets. (A-B) SDS-PAGE separated fractions 1 to 12 obtained by sucrose gradient centrifugation of human (A) and murine (B) platelets probed in IB with specific antibodies against Nbeal2, Thbs1, Crn (Calreticulin), Tuba1c (Tubulin α 1c), and actin (β-actin); for the murine platelets, Crn was not probed but a specific antibody against Vegf (vascular endothelial growth factor) was included. (C-E) Localization of Nbeal2 interactors by IB in the different fractions obtained from platelets of control and Nbeal2^−/− mice, (C) Dock7 in fraction 7 to 8 and fractions 3 to 4, respectively; (D) Sec16a in fraction 1 to 4; (E) Vac14 in fraction 5. (F) Levels of Nbeal2, Dock7, Sec16a, and Vac14 in platelets from control and Nbeal2^−/− mice. (G) Densitometry of the western blot results shown in panel F; total lysates from panel F were performed on platelets of at least 4 (Nbeal2, Sec16a, Vac14) and 8 (Dock7) mice per genotype group. (H) Levels of Dock7 in platelets from GPS cases. For experiments in panels G and H, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, and levels of the other proteins were normalized to GAPDH. Bars represent mean ± SEM. *P value < .05.