FIG. 6.
EDTA-induced NTM processing and nuclear translocation. (A) To demonstrate the intrinsic signaling activity of the NTM subunit, NIH 3T3 cells were cotransfected in triplicate with HES-AB luciferase reporter plasmid, Renilla luciferase control plasmid, and the indicated amounts of pcDNA3 plasmids containing either no cDNA insert, a cDNA insert encoding NTM, or a cDNA insert encoding ΔE. Firefly luciferase activities measured in cell lysates prepared 48 h posttransfection were normalized by using the corresponding internal Renilla luciferase control activities. The fold stimulation values were calculated as the ratio of individual normalized mean HES-AB-luciferase activities to the mean activity in control lysates prepared from cells transfected with empty pcDNA3 vector. In parallel experiments, extracts were prepared from NIH 3T3 cells transfected with either pcDNA3, pcDNA3-NTM, or pcDNA3-ΔE and the Renilla control plasmid. A Western blot normalized for differences in transfection efficiency (based on luciferase levels) was prepared and stained with rabbit anti-IC (inset). (B) ∼105 N1HA cells were treated for 15 min with HBS containing 2.5 mM CaCl2 (C) or 10 mM EDTA (E) at 37°C. The HBS-conditioned medium was harvested (SUP) and analyzed for release of NEC by preparation of immunoprecipitates with anti-HA, while cells were changed back to complete medium (D10) and allowed to recover for up to 8 h. Whole-cell lysates (WCE) prepared at various time points and immunoprecipitates prepared from conditioned media were analyzed on Western blots stained with anti-HA (upper and middle panels) or anti-IC (lower panel). An asterisk denotes a novel anti-IC-cross-reactive polypeptide that appeared in lysates prepared from cells 1 h after exposure to EDTA. (C) A total of 2 × 105 N1HA cells were treated for 15 min with HBS containing either no addition (0) or increasing concentrations of EDTA at 37°C and then allowed to recover in D10 for 1 h. Whole-cell lysates were prepared and analyzed on a Western blot stained with anti-IC. An asterisk denotes a novel anti-IC-cross-reactive polypeptide that appeared in lysates prepared from cells exposed to EDTA. (D) N1HA cells growing on slides were treated with HBS containing 0.5 mM EDTA for 15 min at 37°C, allowed to recover in D10 for 1 h (panel 1), 2 h (panel 2), or 4 h (panel 4) and then fixed and stained with anti-IC and goat anti-rabbit secondary antibody linked to FITC. The immunolocalization of Notch1 polypeptides in cells treated with EDTA was compared to control cells treated with HBS containing 2.5 mM CaCl2 for 15 min at 37°C followed by recovery in D10 for 1 h (panel C).