FIGURE 2:

Comparison of methods to determine surface NPC1L1-3xMyc-GFP and NPC1L1-GFP. Cells transiently expressing NPC1L1-3xMyc-GFP were surface labeled with anti-Myc antibody and analyzed by (bar 1) flow cytometry (αMyc; >7000 cells counted in each of two experiments); bar 2, immunoblotting; bar 3, cell surface biotinylation as in Figure 1. Alternatively, cells expressing (non-Myc-tagged) NPC1L1-GFP transiently (bar 4) or stably (bar 5) were surface labeled with biotin as in Figure 1 and analyzed by immunoblotting. Flow cytometry and immunoblotting were also used to determine the total amount of NPC1L1; shown is the fraction of NPC1L1 detected at the surface. For flow cytometry totals, 5266 or 2430 cells were counted. Stably expressed NPC1L1-GFP was determined in five different experiments in triplicate or quadruplicate (N = 19). For other experiments, the average of triplicate determinations is shown; error bars represent SEM.