Figure 3. NOTCH3 ectodomain interacts with both NOTCH1 and NOTCH4 ectodomains. Interactions between NOTCH3 (WT and mutants) with NOTCH1 or NOTCH4 [41] were tested after cotransfection of plasmid combinations shown. Protein from transfected cells were evaluated by immunoblotting (input) and by immunoprecipitation with V5 monoclonal antibody followed by immunoblotting (IP:V5). Each of the NOTCH3 ectodomain constructs coprecipitated with both V5- tagged NOTCH1 or NOTCH4 proteins, which migrate with apparent molecular weights of .280 kDa and 180 kDa, respectively. All experiments were performed three times. doi:10.1371/journal.pone.0044964.g003 Cellular Effect of CADASIL Mutants of NOTCH3 and 5C). Specifically, decay of the R90C ectodomain was significantly impaired compared to wildtype, while C49Y ectodomain displayed a less severe reduction in clearance. When the wildtype and R90C mutant proteins were cotransfected in cells, the overall clearance of NOTCH3 ectodomains was reduced and displayed an intermediate between wildtype and mutant rates. When C49Y protein was cotransfected with wildtype NOTCH3, the clearance of NOTCH3 proteins adopted the slower clearance rate of the C49Y protein. These results suggest that the C49Y protein (but not the R90C protein) can dominantly influence the clearance rate of coexpressed NOTCH3. NOTCH3 Exerts a Dominant Negative Effects on Notch Signaling Since both WT and mutant NOTCH3 dimerize with other NOTCH proteins and CADASIL mutations in NOTCH caused decreased solubility and clearance of NOTCH3 ectodomains, we hypothesized that increased NOTCH3 could affect Notch signaling. We tested how overexpressed full-length NOTCH3 affects Notch signaling by utilizing a well-established coculture system to measure the effects of expression of wildtype or mutant full-length NOTCH3 on Notch regulated smooth muscle genes. A7R5 rat aortic smooth muscle cells express Notch1 and Notch3 mRNA, and, to a lesser degree, Notch4 transcripts (not shown). SM-MHC, SMA, SM22, and calponin were all upregulated in A7R5 cells by coculture with ligand producing cells (Figure 6). Transfection with full-length Notch3 (WT or mutant) clearly did not increase the expression of these genes; rather, NOTCH3 transfection inhibited ligand dependent in- Figure 4. Detergent solubility and formation of inclusions of NOTCH3 expressed in cell culture. NOTCH3 complexes containing wildtype protein or mutant proteins were tested for detergent solubility. Total protein from cell lines expressing WT or mutant NOTCH3 were sequentially extracted as detailed in methods, using progressively higher strengths of detergents. Cells were grown at low density (A) or in confluent monolayers (B). While WT protein was modestly soluble, the mutant proteins were extractable only in higher detergent concentrations. NOTCH3 protein was detected using antisera from Santa Cruz. The control protein tubulin was readily extractable without detergents. Experiments were performed three times with consistent results. Cell lines were immunostained with the same antisera or with antibodies from R&D, revealing diffuse cell staining with focal accumulations in the perinuclear areas of cells expressing both wildtype and mutant proteins (C). A significant difference in the percentage of cells with perinuclear expression of the protein was notable at low cell density (D) (over 250 cells were counted for each cell line and condition. Results were reproduced three times, and significance was considered p,0.05. doi:10.1371/journal.pone.0044964.g004 PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e44964