Proteolytic processing of wild-type Notch3. (a) Diagram of the Notch3 protein. Bars below the diagram indicate the various regions used as antigens to generate Notch3 antibodies (monoclonal antibodies 5E1, 11A1, and 5G7; polyclonal antibodies BC2 and BC4). TM, transmembrane domain. (b) Western blot analysis of transfected 293T cells and human control arterial tissue. Extracts were prepared from 293T cells transfected with human Notch1 (N1), Notch2 (N2), Notch3 (N3), or pSG5 vector (V). Fragment of a renal artery from a control individual was homogenized and then centrifuged at 16,000 g. The resulting pellet and supernatant were adjusted to 1× SDS-Laemmli buffer. Thirteen micrograms of transfected 293T cells and approximately 100 μg of pellet and supernatant (Sup) from the control artery were run on a 6% SDS-PAGE gel and incubated after transfer with 5E1 (left) and 5G7 (right). Positions of the 280-kDa full-length Notch3 (large black arrow) and the 210-kDa and 97-kDa processing products are indicated (open arrowhead and small arrow, respectively). Notice the weak expression level of Notch3 in arterial tissue, which required more than 1 hour of exposure as opposed to a few seconds for transfected cells. (c) Notch3 210-kDa and 97-kDa processing products are associated. Extracts from 293T cells transfected with Notch3 cDNA (N3) or vector alone (V) were immunoprecipitated with BC2 and BC4 polyclonal antibodies and with preimmune serum (PPI). Precipitated proteins were resolved by SDS-PAGE and immunoblotted with 5E1 and 5G7 antibodies. IP, immunoprecipitation. Migration of molecular-weight markers is shown to the left of each panel.