Figure 4.
The 507* deletion localizes to a BFA-resistant compartment that cofractionates with iLDCVs. (A) PC12 cells stably expressing 507* were incubated without or with 5 μg/ml brefeldin A for 15 min, and then double stained with mouse monoclonal HA antibody and rabbit polyclonal TGN-38 antiserum, followed by anti–mouse antibody conjugated to Cy3 and anti–rabbit antibody conjugated to Cy5. 507* and TGN-38 colocalize in the absence but not the presence of BFA. As indicated by the dispersion of TGN-38, BFA causes fragmentation of the TGN but not the compartment containing 507*. (Insets) Perinuclear staining (arrowheads) at high magnification. Bar, 10 μm. (B) To label SgII in the TGN, cells were incubated for 5 min with 0.5 mCi/ml 35S-sulfate, and then chilled on ice. To allow the labeled SgII to enter iLDCVs, cells were labeled in the same way but incubated at 37°C for an additional 15 min in medium with unlabeled sulfate before harvesting. The resulting post-nuclear supernatants were then separated by velocity sedimentation through 0.3–1.2 M sucrose, and fractions collected from the top of the gradient. Labeled SgII was detected by autoradiography and 507* protein by Western analysis using the monoclonal HA antibody. 507* comigrates with iLDCVs in fractions 2–4 rather than with the TGN in fractions 7–9.