Figure 3.

VIP36-SP-FP colocalizes with cargo blocked early in the secretory pathway at 15°C. The online data show a color overlay and comparison of the two channels for A and B, and an analysis of cross talk. (A) VIP36-SP-FP accumulates with cargo in pre-Golgi transport carriers in the cell periphery. COS cells transiently coexpressing VIP36-SP-CFP and VSVG3-SP-YFP were incubated 6 to 12 h at 39.5°C to accumulate cargo in the ER and then shifted to 15°C for 2 h in the presence of cycloheximide to block cargo in early secretory structures. Cells were then fixed in cold methanol and mounted for high-resolution imaging in PBS. Images are individual, line-interlaced channels of single confocal sections. Inset shows the boxed region enlarged twofold. The reciprocal FP combination (VSVG3-SP-CFP and VIP36-SP-YFP) were imaged with identical results. Bar, 10 μm. (B) High-resolution, close-up view of peripheral endoplasmic reticulum and pre-Golgi structures containing both VIP36-SP-FP and secretory cargo. COS cells transiently coexpressing VIP36-SP-CFP and VSVG3-SP-YFP were treated as in A. Arrowheads indicate punctate structures that accumulate both proteins. N indicates the nucleus. Bar, 5 μm.