Figure 7.

Total and surface levels of LAMP I and II are altered in AP-3 –/– and vimentin –/– fibroblasts. (a and b) Fixed cells were stained for LAMP I and II either in the presence or absence of saponin to assess total (a) or surface (b) staining, respectively. Mean fluorescence intensity was analyzed by flow cytometry, and staining was normalized by subtracting the mean fluorescence values of stainings lacking primary antibodies. Both total and surface staining of LAMPs were increased in AP-3 –/– cells compared with controls. In contrast, both total and surface staining of LAMP I and II was decreased in vimentin –/– cells (n = 3, triplicate asays). (c) Percentage of total LAMP I and II found at the surface was calculated from a and b. The percentage of LAMP at the surface is decreased in cells deficient for AP-3 and increased in cells deficient for vimentin compared with wild-type controls. (d and e) Fibroblasts were surface biotinylated, lysed and biotinylated proteins isolated with avidin beads. Precipitated proteins (lanes 3–6) or 5% of input (lanes 1 and 2) were blotted for either LAMP II or the transferrin receptor. Both total and surface levels of LAMP II are increased in AP-3 –/– fibroblasts (d). Total and surface levels of LAMP II are decreased in vimentin –/– fibroblasts (e). Surface levels of the transferrin receptor remain unchanged. Controls from nonbiotinylated lysates did not result in precipitation of either LAMP II or the transferrin receptor (our unpublished data). (d and e) Lanes 3 and 4 and 5 and 6 are duplicate assays of the biotinylation (n = 2, independent experiments).