APP and Numbs/Numb-like are coexpressed with Notch1 in cortical neurons and are coimmunoprecipitated from adult mouse brain. (A) Representative confocal images (1 μm thick) of cultured cortical neurons immunostained for APP, Numb, Numb-like (all red), and Notch1 (green). (Bar = 15 μm.) (B) Coimmunoprecipitation (IP) of APP and Nbl from total lysates (TL) of adult mouse brain was done by using the α-APP (no. 1736) and α-Nbl antibodies. *, α-Nbl polyclonal antibody crossreacts with all four Numb isoforms. Western blotting (WB) was performed with either the α-Nbl antiserum or the α-APP monoclonal antibody 22C11. The rabbit α-mouse IgG (Rb α-M) antibody was used as a negative control.

Nbl interacts with AID and membrane-tethered APP in vivo. (A) Representative plot of ECFP vs. EYFP. The R16 gate, containing cells with nearly equal ECFP and EYFP intensity, was used to calculate the percentage of cotransfected cells exhibiting FRET. (B) HEK293 cells were transfected with the indicated EYFP-tagged APP/AID constructs (Left) along with ECFP-Nbl constructs (Right). Samples are plotted based on the intensity of the FRET signal (y axis) versus its ECFP or EYFP emission (x axis, left and right column, respectively). Cells in the R11 (ECFP column) or R3 (EYFP column) gates were scored as positive. Percentages are calculated as the number of positive cells divided by the total number of cells, or, in parentheses, the number of cotransfected cells (gate R16). Although both APP and AID interact with Nbl (as indicated by positive FRET), the AID mutants ^12–28AID-EYFP, ^39–55AID-EYFP, and ^44–59AID-EYFP do not exhibit FRET when coexpressed with ECFP-Nbl. In addition, the Nbl deletion mutant ECFP-Nbl^212–603, which lacks the PTB domain, does not interact with AID-EYFP to detectable levels. (C) Effect of the γ-secretase inhibitor DAPT (100 nM) on FRET between Nbl and AID or APP. HEK293 cells were transfected with ECFP-Nbl along with either EYFP-AID or EYFP-APP. Inhibition of γ-secretase activity by DAPT doubles the number of ECFP-Nbl/EYFP-APP transfected cells exhibiting FRET. As expected, DAPT does not affect FRET in ECFP-Nbl/EYFP-AID transfected cells.

AID inhibits NICD signaling in primary cortical neurons. (A) Cultured cortical neurons (5 to 6 days old) were transfected with TP1-luciferase along with NICD, Nbl, APP, AID, or AID^12–28 as indicated. The activity of NICD was inhibited by Nbl, APP, and AID in a dose-dependent manner (0.25, 0.5, and 1 μg/well). No significant change in NICD-dependent transactivation of TP1-luciferase was observed when NICD was cotransfected with AID^12–28. Significance was determined by using a two-tailed Student's t test (*, P < 0.05; **, P ≤ 0.01). (B) Representative confocal images (1 μm thick) of cultured cortical neurons transiently transfected with AID-EYFP, AID^12–28-EYFP, APP-EYFP, and EYFP-Nbl fusion constructs (shown in green). Neuronal DNA was stained with propidium iodide (shown in red). (Bar = 20 μm.)

AID interacts with Nbl and Numbs. (A) HEK293 cells were transfected with either AID-ECFP or ECFP along with Flag-Numb p72 (p72, Upper), Flag-Numb p71 (p71, Upper middle), Flag-Numb p66 (p66, Lower middle), or Flag-Numb p65 (p65, Lower). Cell lysates were coimmunoprecipitated (IP) with either α-Flag or α-Living Colors (α-LC), which recognizes ECFP- and EYFP-tagged proteins. Expression of transfected constructs in total lysates (TL) and immunoprecipitates (IP) also were analyzed by Western blot (WB) with the α-Nbl or α-LC antibodies. p72ΔN, p71ΔN, p66ΔN, and p65ΔN are recognized by the α-Nbl antisera, which is specific for the COOH-terminal region of Numb/Nbl, but are not immunoprecipitated by the α-Flag antibody, which is specific for the Flag epitope fused at the NH₂ terminus of these proteins. Therefore, they represent degradation products lacking the NH₂-terminal portion and part of the PTB domain, which do not interact with AID-ECFP. (B) HEK293 cells were transfected with AID-ECFP or ECFP along with either Flag-Nbl (lanes 1–6) or the unrelated protein Flag-AIP (lanes 7–12). Lysates were immunoprecipitated with either the α-Flag or α-LC antibodies as indicated. Lysates and immunoprecipitates were analyzed by WB with either the α-Flag, α-LC, or α-Nbl antibodies as indicated. NblΔN is a degradation product of Nbl lacking the NH₂-terminal PTB domain and does not interact with AID-ECFP (see above for explanation). * indicates degradation products of AID fusion proteins containing ECFP alone, which does not bind Nbl. (C) HEK293 cells were transfected with Flag-Nbl along with EYFP-tagged deletion construction of AID as indicated. Lysates were immunoprecipitated with α-Flag and analyzed by WB with either α-Nbl or α-LC as indicated. AID mutant numbering is based on the AID59 peptide sequence. (D) HEK 293 cells were transfected with either AID-EYFP or ^12–28AID-EYFP along with Flag tagged deletion constructs of Nbl as indicated. Lysates were immunoprecipitated with α-Flag and analyzed by WB with either α-Flag or α-LC as indicated. ** indicates the signal from the α-Flag heavy chain. The difference in molecular mass between AID-EYFP and mutant forms of AID-EYFP, which can be appreciated in (C), is not apparent in (D), because the proteins were separated less extensively by SDS/PAGE.

Processing of APP and release of AID inhibits Notch signaling. (A) HeLa cells were transfected with 4xCBF1-luciferase (CBF-luc) along with the indicated Notch1 and Nbl constructs. Nbl inhibits the transactivation of CBF-luc by both NICD and NΔE. (B) Western blotting (WB) with the α-Nbl antiserum shows expression of Nbl and/or Numb proteins in HeLa cells (Top). WB with α-APP (22C11) shows induction of APP by Dox in APP-HeLa Tet-on cells (Middle). An α-PARP antibody (2C10) was used to normalize for protein loading (Bottom). (C and D) APP-HeLa Tet-on cells were transfected with CBF-luc along with (C) NICD or (D) NΔE. Some samples were treated with Dox to induce APP expression and/or with the γ-secretase inhibitor DAPT (100 nM). The decrease in NICD activity by APP induction is inhibited by DAPT (C). DAPT treatment significantly reduces NΔE activity in HeLa Tet-on cells (D). (E–H) HeLa cells were transfected with CBF-luc along NICD, NΔE, AID, or mutant forms of AID as indicated. AID inhibits the activation of CBF-luc by both NICD and NΔE (E). The inhibition is dose-dependent (F) and correlates with the ability of AID to interact with Nbl (G). (H) HeLa cells were transfected with either CBF-luc or GAL4-luciferase (GAL4-luc) reporter genes along with APPCT-Gal4 in the presence or absence of cotransfected Fe65. APPCT-Gal4 reduces NICD activity but enhances Fe65 transactivation. Significance was determined by using a two-tailed Student's t test (*, P < 0.05; **, P ≤ 0 01; ***, P ≤ 0.001).