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. 2000 Mar 1;105(5):597–605. doi: 10.1172/JCI8047

Figure 4.

Figure 4

Selective accumulation of the 210-kDa cleavage product in CADASIL patients. (a and b) Western blot analysis of brain extracts from CADASIL and control individuals with antibodies raised against Notch3 extracellular domain. (a) Brain fragments were homogenized and centrifuged at 16,000 g; the resulting pellet and supernatant were adjusted to 1× SDS-Laemmli buffer. Approximately 100 μg of extracts were loaded per lane on a 6% SDS-PAGE gel, and were immunoblotted with the 5E1 antibody. Blots were stained with Ponceau S to confirm that equal amounts of proteins were loaded. Left panel, brain extracts (supernatant and pellet) from CADASIL patient 1 and control individuals 2 and 5. Middle panel, brain extract (pellet) from CADASIL patient 1 and extract from 293T cells transfected with Notch3 cDNA (N3). Right panel, brain extracts (pellet) from CADASIL patients 1, 8, and 9. Position of the 210-kDa Notch3 protein (open arrowhead) detected in CADASIL brain extracts is indicated. The asterisk marks a band of unknown significance (cross-reacting material or, most likely, post-lysis degradation product). (b) Brain extracts from CADASIL patient 1 (pellet) and control individual 2 (pellet), and extracts from 293T cells transfected with Notch3 cDNA (N3) were run on a 6% SDS-PAGE gel and immunoblotted with the 11A1 antibody (left panel) or without primary antibody (–) (right panel). (c) The 97-kDa Notch3 protein is not detected in either CADASIL or control brains. Whole lysates were prepared from brains of CADASIL patient 1 and control individual 5, and from a renal artery of a control subject. Approximately 100 μg of each extract was loaded on a 6% SDS-PAGE gel and immunoblotted with the 5G7 antibody (bottom) and the 5E1 antibody (top). Positions of the 210-kDa and the 97-kDa processing products are indicated (open arrowhead and small arrow, respectively). (d) Western blot analysis of extracts from CADASIL and control arterial tissues. Whole lysates were prepared from mesenteric arteries of CADASIL patient 1 and a renal artery of a control subject. Extracts were resolved on a 6% SDS-PAGE gel and immunoblotted with the 5G7 antibody (right) and the 5E1 antibody (left). The asterisk marks a band of unknown significance (cross-reacting material or, most likely, a post-lysis degradation product). Whole lysates were used in experiments shown in c and d to avoid missing any soluble Notch3 intracellular fragment. (e) Western blot analysis of extracts from CADASIL and control brains and from control arterial tissue under reducing and nonreducing conditions. Extracts from CADASIL and control brains and from a control artery were solubilized in SDS-Laemmli buffer with or without βME and were resolved on a 6% SDS-PAGE gel. Both stacking (bracket) and separating gels were immunoblotted with the 5E1 antibody. Under reducing conditions (βME+), the 210-kDa Notch3 protein is detected in both control artery and CADASIL brain (open arrowhead). Under nonreducing conditions (βME–), no band is detected in the CADASIL brain; a protein of approximately 280 kDa (large open arrow) is detected in the control artery. This protein probably corresponds to the associated extracellular and intracellular domains. Cross-reacting material is caught at the transition between the stacking and separating gels.