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. 2007 Jul 30;178(3):465–476. doi: 10.1083/jcb.200702009

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Copyright © 2007, The Rockefeller University Press

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Figure 6. — Localization of DLL1 and DLL3 proteins. (A) Western blot analysis of cell lysates (input) and streptavidin-immunoprecipitated protein after surface biotinylation (IP). CHO cells stably expressing DLL3 (b and c) at amounts similar to cells expressing DLL1 (a) present significantly less DLL3 on the surface. L cells (mouse fibroblast cell line) coexpressing DLL3flag at significantly higher levels than DLL1HA (compare input lanes d and g) present DLL1 efficiently on the surface but not DLL3 (compare IP lanes d and g). CHO cells coexpressing DLL3HA and DLL1flag (compare input lanes e and f with h and i) present DLL1 efficiently on the surface but DLL3 only in trace amounts (compare IP lanes e and f with h and i). (B) Detection of DLL1 and DLL3 by immunofluorescence. (a–e) Localization of DLL1 and DLL3 in overexpressing CHO cells. CHO cells expressing DLL1 (a and c) show a clear cell surface staining, whereas DLL3 (b and d) is detected almost exclusively inside the cell. DLL1 and DLL3 colocalize only in some vesicular structures (e, arrowheads) but not significantly at the membrane. (f–j) Localization of DLL1 and DLL3 in D. melanogaster wing disc cells. (f) Overview of a wing disc stained for the apical cell membrane marker aPKC and DLL3flag, and Hoechst staining to visualize nuclei. (g and h) Confocal images of two opposed apical cell membranes (red) of an epithelial fold in panel f. DLL3 is found in intracellular granules or vesicles. (i and j) Confocal images of two opposed apical cell membranes (red) of a wing disc stained for aPKC and DLL1flag. DLL1 outlines cell membranes and colocalizes at the apical membrane with aPKC (j, arrowheads). (k–v) Immunofluorescent detection of DLL1 and DLL3 in PSM cells of E9.5 embryos. Endogenous DLL1 is present at the surface (k) and colocalizes with the membrane (m) and in vesicular structures with the cis-Golgi marker GM130 (s). DLL3 does not localize to the membrane (n) and does not colocalize with anti-pancadherin staining (p) but is detected in vesicular structures in the cytoplasm (t), mostly overlapping with GM130 (v). Bars, 10 μm.