FIG 2.

Notch2 and NOTCH3 proteolytic processing is regulated by presenilin-1 or -2. (A) Immunoblots showing expression of endogenous Notch2 in lysates from mouse embryonic fibroblasts (mEF) with double knockout for presenilin-1 and -2 (PS1/2dKO) or reconstituted with presenilin-1 (PSEN1) or 2 (PSEN2) (top panel), in coculture with OP9-J1 cells (second panel) or with coated Dll4-Fc molecules (third panel) in the absence or presence of GSI and GSI/BB94 (fourth panel). Immunoblots for presenilin-1 and -2 proteins (PS-1 and PS-2) show reconstitution of the respective presenilin, as indicated. ARNT (aryl hydrocarbon receptor nuclear translocator) served as a loading control (Ctrl). PS1/2dKO cells were unable to S3 process Notch2, indicated by accumulated N2EXT fragments after stimulation by either J1 or recombinant Dll4-Fc molecules. In the cells with PSEN1 or PSEN2, N2EXT fragments were observed only in the presence of GSI. Treatment with BB94 abrogates formation of N2EXT, indicating that the formation of N2EXT is dependent on metalloproteases. (B) Dual Notch (CSL) luciferase reporter gene activity, corrected for Renilla luciferase expression, in either PS1/2dKO cells or cells reconstituted with presenilin-1 (PS-1) or -2 (PS-2). In response to coculture with Jagged1-expressing cells, a γ-secretase-dependent increased transcriptional activation was observed only in cells reconstituted with either PS-1 or PS-2 and not in PS1/2dKO cells. All values were normalized to DMSO-treated PS1/2dKO cells and arbitrarily set to 1. Measurements correspond to at least two experiments in triplicate and are displayed as relative light units (RLU). Error bars represent means ± standard deviations. (C) Coculture experiment of NOTCH3-HA-expressing PS1/2dKO cells reconstituted with either PSEN1 or PSEN2, Adam10KO, and Adam17KO cells with Dll1-expressing cells compared to OP9 parental cells. HA immunoblots show diminished levels of TMIC after stimulation with Dll1. (Top three panels) N3EXT fragments accumulate in the absence of presenilin proteins, whereas in the presence of either PSEN1 or PSEN2, N3EXT accumulation was observed only when treated with GSI. (Bottom two panels) N3EXT fragments were absent in Adam10KO cells but not in Adam17KO cells after stimulation by Dll1 in the presence of GSI. (D) (Top) HA immunoblotting of cell lysates of nontransfected PS1/2dKO and PSEN1 reconstituted cells or transfected with an EGF repeat and PEST domain NOTCH3-HA deletion construct (hN3ΔE-ΔPEST) in the absence or presence of GSI. N3EXT accumulation was observed in hNOTCH3ΔE-ΔPEST-expressing PS1/2dKO cells without ligand stimulation. In PS1/2dKO cells reconstituted with PSEN1, N3EXT accumulation could be observed only in the presence of GSI. Furthermore, truncated N3ICD fragments were observed in PS1/2dKO cells reconstituted with PSEN1, which were absent in the presence of GSI. (Bottom) Immunoblot showing PS-1 expression with a nonspecific band (ns). Molecular masses are indicated.