Ultrastructure and protein analysis of MKs. (A-D) Ultrastructure of culture-derived MKs from control (A) and Nbeal2^−/− (B) mice at low magnification (A-B) and high magnification (C-D), respectively. MVB precursors of α-granules are present in both control and Nbeal2^−/− MKs. (E-F) Ultrastructure of bone marrow MKs in control and Nbeal2^−/− animals at low magnification. (G-J) Ultrastructure of bone marrow MKs from control and Nbeal2^−/− mice at high magnification. A well-defined demarcation membrane system with the presence of platelet territories and α-granules (AG) in control cells (G, I). The demarcation membrane system in Nbeal2^−/− MKs is not well-defined (H,J), however, α-granules (AG) are present. (K) Low magnification of a proplatelet-forming MK from Nbeal2^−/− bone marrow. (L) High magnification of the circled area in (K) highlights the presence of AG inside the emerging platelet fields, and vacuoles (V) within the MK cytoplasm. (M) Representative immunoblots of α-granule proteins (VWF) (THBS1 and PF4) in control and Nbeal2^−/− MKs. CD63 (LAMP-3), a lysosomal/dense granule marker, and β-actin and glyceraldehyde-3-phosphate dehydrogenase included as loading controls. Below, graphs show the densitometry analysis performed using ImageJ (n = 3). Bars represent mean ± standard error of the mean. NS, nonsignificant. Bar represents 2 μm (A-B,E-F,K) and 500 nm (C-D,G-J,L). Representative images were selected from 5 different mice per group.