Figure 7.

Localization of chimeric DLL1 and DLL3 proteins. (A) Schematic representation of chimeric proteins containing the ICD of DLL1 (left) or DLL3 (right) arranged according to the extent of extracellular DLL1 sequences (top to bottom). Gray parts and red parts indicate DLL1 and DLL3 sequences, respectively. White filling indicates DSL domains, light gray or red shading indicates TMs, and filled boxes indicate EGF repeats. (B) Confocal images of CHO cells stably expressing chimeric ligands and stained by indirect immunofluorescence. Similar to DLL1 (a) and DLL1 lacking the ICD (b), chimeric ligands that contained the TM-ICD of DLL1 and the DLL1 N-terminal portion including the DSL domain were detected on the cell surface (c–e), in addition to some variable intracellular expression. Presence of the DLL1 N terminus alone was not sufficient to direct detectable surface expression (f), similar to the extracellular domain of DLL3 fused to DLL1 TM-ICD (g). Surface presentation of a chimera containing the DLL1 extracellular domain juxtaposed to the DLL3 TM-ICD (h). Chimeras that contain the DLL1 N-terminal portion including the DSL domain, and the DLL3 TM in the context of juxtaposed DLL3 intra- and extracellular sequences were retained intracellularly (i and j). Intracellular localization of DLL3 with the N terminus replaced by the corresponding DLL1 sequence, and of DLL3, respectively (k and l). A–C, G, and H refer to chimeras shown in Fig. 5 A and I–M to additional ones. Chimera H (b) was detected with anti-DLL1 and all other chimeras with anti-flag antibodies. Bar, 10 μm.