1. A Novel Proteolytic Cleavage Involved in Notch Signaling
Christel Brou, Frédérique Logeat, Neetu Gupta, Christine Bessia, Odile LeBail, John R Doedens, Ana Cumano, Pascal Roux, Roy A Black, Alain Israël 
Molecular Cell 
Volume 5, Issue 2, Pages (February 2000)
DOI: /S (00)

2. Figure 1 Pulse-Chase Analysis of ΔE-GALVP16 Constructs
(A) Schematic map of the ΔE-GALVP16 substrate used in (B). SP, signal peptide; TM domain, transmembrane domain; VSV, VSV epitope tag. The S2 and S3 sites are indicated, and the amino acid coordinates are from murine Notch1.
(B) 293T cells were transfected with plasmids encoding ΔE-GALVP16 wild type (lanes 1–3), carrying the AV→VH mutation at the S2 site (lanes 4–6), or carrying the GCGV→LLFF mutation (R. Kopan, personal communication), at the S3 site (lanes 7–9). After 24 hr, cells were pulsed with [35S]Met for 15 min, then directly extracted (lanes 1, 4, and 7) or chased for 3 hr (lanes 2, 3, 5, 6, 8, and 9). MG132 (50 μM, lanes 3, 6, and 9) was applied continuously 1 hr before and during the pulse chase. Cells extracts were immunoprecipitated with anti-VSV antibody (P5D4). Immunoprecipitated proteins were eluted and analyzed on a 8% SDS-PAGE. ΔE-GALVP16, P2, and P3 respectively indicate the uncut substrate and the P2 and P3 processing products.
Molecular Cell 2000 5, DOI: ( /S (00) )

3. Figure 2 In Vitro Cleavage at the S2 Site
(A) Schematic map of the FuGALVP16 substrate used in (B). The sequence of the region surrounding sites S2 and S3 is indicated. The transmembrane domain is boxed.
(B) The substrates FuGALVP16, either wild type (lanes 1–3) or mutated at the S2 site (lanes 4–6), containing a Notch fragment (amino acids 1655–1809) in frame with a VSV epitope and the chimeric protein GALVP16, were in vitro transcribed and translated in the presence of [35S]Met. An aliquot was incubated without (lanes 1 and 4) or with (lanes 2, 3, 5, and 6) HeLa cells membranes. DTT was added to 1 mM when indicated (lanes 3 and 6). The products were analyzed on an 8% SDS-PAGE. UC and C represent the substrate and its P2 processing product, respectively.
Molecular Cell 2000 5, DOI: ( /S (00) )

4. Figure 4
The Metalloprotease Kuz and the S2 Processing Activity Can Be Chromatographically Separated
A HeLa cells membrane preparation was fractionated on Q-Sepharose and RED-TSK columns, and the fractions were tested in parallel for the presence of the Kuz protein by Western blotting (upper panel) and for their capacity to cleave ΔE-GALVP16 at the S2 site in vitro (lower panel). The two lanes for each fraction in the lower panel correspond to 1 and 10 μl of fraction per assay. The apparent molecular weight of Kuz is about 60 kDa, as previously described (Howard et al. 1996).
Molecular Cell 2000 5, DOI: ( /S (00) )

5. Figure 6 TACE Accounts for the S2 Activity
Fractions derived from the purification of the S2 activity (Concanavalin A–eluted material, lanes 2 in [A], [B], and [C], or MonoQ-eluting fraction in lanes 3 of [A] and [B]) or from culture supernatants of cells overexpressing recombinant forms of TACE (TACE cat only contains the catalytic domain of the enzyme: lanes 5 in [A] and [B], two differents doses in lanes 4 and 5 of [C]; TACE EC contains the extracellular domain of the enzyme except for the prodomain: lanes 6 in [A] and [B], lanes 6 and 7 in [C], increasing doses) were tested in vitro using different substrates. The reactions were performed in the same conditions for the three substrates: ΔE-GALVP16 wild type in [A], the AV→ED mutant in [B], or pro-TNFα in [C]. UC and C represent the substrate and its processing product, respectively. Extracts derived from TACE+/+ (lanes 7 in [A] and [B]) or TACE−/− fibroblasts (lanes 8 in [A] and [B]) were tested in parallel.
Molecular Cell 2000 5, DOI: ( /S (00) )

6. Figure 3 The Second Processing Event Can Occur In Vivo
(A) Total extracts were prepared from Jurkat (lane 1) or C12D cells (lanes 2 and 3) and analyzed by Western blotting using P1, an antibody recognizing the extracellular region of TACE (the results concerning pro-TACE were confirmed using an antiserum directed against the prodomain of TACE [data not shown]). When indicated, the cells were treated for 3 hr with glucose oxidase (GO, 0.02 U/ml) before extracts were prepared. The positions of the precursor form (pro-TACE) and the mature form (TACE) are indicated on the right. Ju, Jurkat. A molecular weight marker (kDa) is shown on the left.
(B) The extracts were analyzed using an antiserum recognizing the intracellular region of the Notch1 molecule (Logeat et al. 1998). GO, glucose oxidase. The p300 Notch1 precursor, the p120 furin-generated product, and the P3 product are indicated on the right.
(C) An analysis similar to (B) was performed, except that a longer gel with a higher concentration of acrylamide was used, to focus on the 100–120 kDa region. Jurkat (lanes 1–3) or C12D cells (lanes 4–9) were treated for 3 hr with glucose oxidase (0.02 U/ml), 1,10 o-phenanthroline (5 mM), and/or MG132 (50 μM) as indicated below the lanes. The P1 (p120), P2, and P3 products are indicated on the right. n.s. represents a nonspecific band.
(D) Nuclear extracts from C12D cells, treated or not with glucose oxidase as indicated, were analyzed by Western blotting with the anti-Notch antibody (top) or an anti-CSL antibody as a control for extraction of nuclear proteins (bottom). The P3 processing product and CSL protein are indicated on the right.
(E) Total RNA extracted from untreated or glucose oxidase treated Jurkat or C12D cells, as indicated, was analyzed by Northern blotting using a HES-1 probe (top) or an S26 probe for normalization. Quantification by Phosphorimaging and normalization with respect to S26 showed a reproducible 2-fold increase in HES-1 expression in glucose oxidase-treated C12D cells and no increase in Jurkat cells.
Molecular Cell 2000 5, DOI: ( /S (00) )

7. Figure 5
TACE and the S2 Activity Coelute throughout the Purification Procedure
S2 activity was purified from HeLa cells by successive fractionation onto Q-Sepharose (first three lanes), RED-TSK (three following lanes), Concanavalin A-Sepharose, RED-TSK, and finally onto a MonoQ column (last nine lanes) (see Experimental Procedures). The fractions were tested by Western blotting with anti-TACE antibody (M222) or by in vitro assay on ΔE-GALVP16. The plus signs indicate that the fraction contains the S2 activity, the size being proportional to the activity. It should be noted that the peak of activity detected in vitro correlates with the highest concentration of TACE. The apparent molecular weight of TACE is about 80 kDa, as described (Black et al. 1997).
Molecular Cell 2000 5, DOI: ( /S (00) )

8. Figure 7
Inhibition of PMA-Induced Differentiation of DRM Cells Is Dependent on the Presence of TACE
(A) TACE-reconstituted bone marrow–derived monocytic precursor cells (DRM-5) (obtained as described in Experimental Procedures by infecting TACE−/− cells with a retrovirus expressing murine TACE) were treated with 100 ng/ml PMA for 0 (panel 1), 24 (panels 2 and 4), or 48 hr (panels 3 and 5) in the absence (panels 2 and 3) or presence (panels 4 and 5) of 50 μM MW167. (B) TACE−/− DRM cells were treated with 100 ng/ml PMA for 0 (panel 1), 24 (panel 2), or 48 hr (panels 3 and 4) in the absence (panels 2 and 3) or presence (panel 4) of 50 μM MW167. The cells were then fixed and stained with May-Grünwald-Giemsa as described in the Experimental Procedures. Red arrows indicate blast precursor cells (small cells with a dark nucleus and almost no visible cytoplasm), while green arrows indicate differentiated cells (larger cells, pale pink, nucleus poorly visible). (C) Western analysis of Jagged1 in TACE-reconstituted (TACE+/+) or TACE−/− DRM cells treated or not with PMA for 24 or 48 hr, as indicated.
Molecular Cell 2000 5, DOI: ( /S (00) )