Identification of the EC cleavage site, generation of a Dll1 mutant for this cleavage, and processing of Dll1 in Kuz–/– cells. (A) Alignment of mDll1 with Dll1-D8 and Dll1-Apa mutants and Delta orthologs, in the juxtamembrane region (amino acids 516–545 of mDll1). The two cleavage sites identified by Mishra-Gorur et al. (19) in Drosophila Delta and the identified cleavage site of Dll1 are indicated. (B) Identification of the cleavage site. HEK293T cells were transfected with Delta-Myc₆. After 24 h, cells were labeled with [³H]Leu or [³⁵S]Met. Whole-cell extracts were immunoprecipitated with anti-Myc antibody. By radio sequencing of a ³⁵S peak was detected at cycle 1 and a ³H peak at cycle 19. The corresponding amino acid sequence is shown (Lower). (C) Analysis of the noncleavable Dll1-D8 and Dll1–Apa mutants. HEK293T cells were transfected with WT Dll1 (lane 2), Dll1-D8 (lane 3), or Dll1-Apa (lane 4), and whole-cell extracts were blotted with Dll1 antiserum. (D) Proteolytic cleavage of Dll1 is reduced in Kuz–/– cells. Kuz +/+ (lanes 1 and 2) and –/– (lanes 3 and 4) MEFs were infected with a retrovirus encoding GFP alone (MIG) or V-F-Dll1-internal ribosomal entry site-GFP (V-F-Dll1). GFP-enriched pools of cells were analyzed by immunoblotting of whole-cell extracts by using anti-Flag antibody. Equal protein loading was controlled by using an anti-GFP antibody (the level of GFP being correlated with the level of Dll1 expression).

Dll1 is cleaved by a presenilin-dependent γ-secretase activity. PS1 +/+ and –/– MEFs were infected by a retrovirus encoding GFP alone (–) or V-F-Dll1-internal ribosomal entry site-GFP (+). (A) Dll1^IC release is inhibited by MW167. GFP-enriched pools of PS1+/+ cells were analyzed by immunoprecipitation using the anti-Flag antibody followed by immunoblotting with the Dll1 antiserum. (Upper) Short exposure. (Lower, corresponding to the boxed region) Long exposure. Dll1^IC indicates the position of the soluble IC fragment. In lane 3, PS1+/+ cells were treated for 15 h in the presence of the γ-secretase inhibitor MW167 (MW) before lysis. (B) Absence of Dll1^IC release in PS1–/– cells. A similar experiment to lanes 1 and 2 of A was performed in parallel in PS1+/+ (lanes 4 and 5) and PS1–/– (lane 6) cells.

Murine Dll1 undergoes a constitutive ectodomain shedding. (A) Schematic map of the tagged Dll1 molecule used in B and C. (B and C) Pulse– chase analysis of Dll1. HEK293T cells were transfected with V-F-Dll1. After 24 h the cells were pulsed with [³⁵S]Met (t0) for 20 min and chased for 0.5, 1, 2, 4, and 6 h. Cell extracts were immunoprecipitated with anti-VSV (B) or anti-Flag (C) antibodies. Culture media were immunoprecipitated with anti-VSV antibody (B Lower). Dll1 indicates full-length Dll1, Dll1^TMIC is the membrane-associated processing product, and Dll1^EC is the shedding product. Molecular mass markers are indicated on the left.

Full-length Dll1 and its proteolytic fragment Dll1^TMIC are present in distinct complexes. (A) Gel filtration analysis of extracts from 293T cells transfected with Dll1. Whole-cell extracts were fractionated through a Superose 6 column. Fractions were analyzed by immunoblotting, using Dll1 antiserum (Upper), followed by PS1 antiserum (Lower) after membrane stripping. Positions of full-length Dll1, Dll1^TMIC, and the N-terminal processing product of PS1 (PS1 NTF) are indicated on the right. (B) Dll1 interacts with PS1. HEK293T cells were transfected with plasmids encoding V-F-Dll1 and/or PS1 as indicated. Whole-cell extracts (WE; lane 1) and anti-PS1 immunoprecipitates (IP anti-PS1; lanes 2–4) were analyzed on SDS/PAGE and immunoblotted with an anti-Flag antibody. The position of Igs is indicated on the right.

Generation of Dll1^TMIC is a preliminary requirement for γ-secretase cleavage to occur. (A) Dll1-Apa and Dll1-D8 do not undergo γ-secretase cleavage. HEK293T cells were transfected with V-F-Dll1 (lanes 1, 4, and 7), V-F-Dll1-Apa (lanes 2, 5, and 8), and V-F-Dll1-D8 (lanes 3, 6, and 9) and treated with lactacystin for 3 h. Membrane, cytosolic, and nuclear extracts were prepared and analyzed by immunoblotting using the anti-Flag antibody, followed by immunoblotting with anti-β-tubulin (a cytosolic marker) and anti-CSL (a nuclear marker) antibodies as a control for the purity of the fractions and equal protein loading. (B) Dll1^IC localized to the nucleus. HeLa cells were transfected with Dll1^IC-V5 and treated with lactacystin for 3 h. Cells were stained with the anti-V5 antibody (Left) and Hoechst for nuclear staining (Center). Light blue in the merged image (Right) indicates colocalization.