Fig. 6. Overexpression of DN Rab7 blocks delivery of 
-mannosidase and LmpA to phagosomes. In (A), vesicles of the endo/lysosomal system were purified by pulsing control cells with iron dextran for 60 minutes and purifying iron dextran-containing vesicles using a magnetic column (see Materials and Methods). Western blots of cell homogenate (H) and endo/lysosomal vesicles (E/L) were decorated with antibodies to LmpA (lysosomal integral membrane protein), 100 kDa and 41 kDa subunits of the V-ATPase (100 kDa S.U. and 41 kDa S.U.), -mannosidase (-mann.), cathepsin D (Cath. D), cysteine proteinase P36 (CPp36) and Rab7 as described in Materials and Methods. In (B), phagosomes were purified as described in Materials and Methods from cells pulsed with latex beads for 5 minutes and chased for the indicated periods of time. Western blots were performed as in A. Bottom strip, cysteine proteinase activity of phagosomal fractions. In (C), phagosomes were purified from control cells (x4) and cells overexpressing DN Rab7 (7TN) pulsed with latex beads for 15 minutes. Western blots were performed as in A. The positions of marker proteins (kDa) are shown.
