Rab27b is hypoprenylated in gm/gm MKs and expressed abundantly in late stages of normal MK maturation. (A) In vitro prenylation assay shows that a limited number of Rab proteins is hypoprenylated in gm/gm MKs. Cytosolic lysates were incubated in the presence of recombinant RGGT, Rab escort protein 1 (REP-1), and [³H]geranylgeranyl pyrophosphate as the lipid donor, followed by gel electrophoresis and autoradiography. (B) Immunoblot of postnuclear supernatant extracts (PNS; 25 μg) or the soluble (S₁₀₀) and membrane-associated (P₁₀₀) fractions from +/gm and gm/gm MKs using Rab27a (4B12), Rab27b (S086), or calnexin (loading control) antibodies. The results indicate hypoprenylation of Rab27a and Rab27b in gm/gm MKs. (C) Immunoblot analysis of +/ash and ash/ash MKs using 4B12, R27B1 (anti-Rab27b), or an antibody (N688) that reacts with both Rab27a and Rab27b. Total Rab27 levels in ash/ash MKs are similar to control cells despite absence of Rab27a; this implies that Rab27b is the predominant subfamily member present in maturing MKs. (D) Immunoblot of MK cell populations enriched for different stages of maturation (Im indicates immature MKs on fetal liver culture day 3; M, mature MKs with few or no proplatelets on culture day 4; and M+PP, predominantly terminally differentiated proplatelet-producing MKs on culture day 6) with antibodies specific for known platelet-expressed Rab proteins or GAPDH. Relative levels of the chemiluminescence signal were quantified digitally and are represented in the right panel. Levels of Rab27b increase the most dramatically with MK maturation and proplatelet formation.