Soluble and membrane-bound α-granule protein deficiencies in ARC platelets and the presence of VPS33B in normal fibroblasts and megakaryocytes. Immunoblots comparing megakaryocyte (MK) and platelet (PLT) or fibroblast whole-cell lysates from ARC and normal (N) neonatal and adult sources as indicated for each lane. Actin was visualized as a protein concentration loading control. (A) Significantly decreased β-TG was observed in ARC neonatal platelets (lane 1) compared with normal neonatal (lanes 2, 3) and adult (lane 4) platelets (reduced 15% SDS-PAGE; β-TG and actin were probed and developed simultaneously). (B) ARC platelets contained undetectable amounts of TSP-1 (lane 1) compared with normal neonatal platelets (lane 2, reduced 10% SDS-PAGE). (C) Fibrinogen was undetectable in ARC platelets (lane 1) despite its normal presence in neonatal control platelets (lane 2, nonreduced 9% SDS-PAGE) and ARC plasma (not shown). (D) The α-granule membrane-containing protein P-selectin was virtually undetectable in ARC platelets (lane 1) when compared with normal neonatal (lanes 2, 3) and adult (lane 4) platelets (reduced 8% SDS-PAGE, stripped VWF blot from Figure 3C). (E) Affinity-purified polyclonal anti–human VPS33B detected VPS33B in normal neonatal fibroblasts (lane 2) and normal adult megakaryocytes (lane 3), but not in ARC neonatal fibroblasts (lane 1) or normal adult control platelets (lane 4). Actin immunostaining is shown on the same blot.