Cellular Effect of CADASIL Mutants of NOTCH3 Figure 2. Interactions between wild-type and R90C and C49Y mutants of NOTCH3. (A–C) NOTCH3-NOTCH3 self-association is mediated by the extracellular domain. 293 cells were transiently co-transfected with cDNAs expressing the NOTCH3 extracellular domain tagged with HA (A, B, and C were transfected with WT, R90C, and C49Y, respectively) and cotransfected with myc tagged NOTCH3 ectodomain constructs as shown. The proteins were extracted after 40,48 hours and immunoprecipitated with HA monoclonal antibody. Co-immunoprecipitates of cells cotransfected with plasmid combinations shown were analyzed by immunoblotting (IB). Quantification of protein bands from four independent experiments failed to show a significant difference in IP complexes with any of the NOTCH3 combinations. (D) Effect of divalent cations on stability of NOTCH3 homophilic interactions and the stability of NOTCH3 homophilic complexes. Coimmunoprecipitation was performed using the HA antibody on lysates of cells transfected with plasmid combinations shown on the right column. Immunoprecipitates were washed extensively with buffers containing calcium or magnesium, and then analyzed by western blotting. All of the HA and myc tagged proteins run on SDS gels at an apparent molecular weight of 175 kDa. We verified the finding of numerous groups that common epitope tags do not mediate protein interaction [64,65,66] (not shown). All experiments were performed more than 6 times. doi:10.1371/journal.pone.0044964.g002 the stability of NOTCH3 protein complexes (Figure 2D), regard- less of the presence of mutant NOTCH3 in complexes. NOTCH1 and NOTCH4 are also expressed in blood vessels and A7R5 smooth muscle cells [53,54,55,56] (data not shown). The ectodomains of these isoforms of NOTCH, like NOTCH3, are composed of a large number of EGF-like domain repeats. In cotransfection assays, we also found that wildtype and mutant NOTCH3 formed stable complexes with NOTCH1 and NOTCH4 ectodomains (Figure 3). CADASIL Mutations Decrease Solubility of NOTCH3 in Cell Lines We next assessed the solubility of full-length NOTCH3 wild type and mutant protein complexes from stably transfected cell lines. Full-length wildtype NOTCH3 complexes were found primarily in low concentration detergent fractions when expressed in cells plated at low density (Figure 4A). In contrast, full-length mutant NOTCH3 complexes were completely insoluble in detergent or required high concentrations of detergent for solubilization. At higher density of plating, there was a modest increase in NOTCH3 solubility (Figure 4B). Cell lines stained for NOTCH3 demonstrated expression in the cell body and perinuclear regions (likely in the Golgi complex), reminiscent of that seen by other investigators [57,58,59]. Perinuclear foci were similar in wildtype and mutant full-length NOTCH3 expressing lines (Figures 4C and 4D). Plating at high cell density strongly reduced perinuclear accumulations of NOTCH3. NOTCH3 Mutations Delay Protein Clearance from Cells We next tested the stability of NOTCH3 ectodomain in live cells. We fused wildtype and mutant NOTCH3 ectodomains to firefly luciferase (Figure 1), which allowed consistent and sensitive measurement of protein levels. Cells expressing these fusion proteins were treated with cycloheximide to inhibit further protein synthesis. At the end of specified periods after halting protein synthesis, cell lysates were analyzed for levels of cell-associated luciferase expression. All proteins were expressed at similar activity levels prior to the addition of cycloheximide (Figure 5A). As expected, all proteins displayed a progressive decrease in protein expression over time after cycloheximide treatment. However, cells cleared the wildtype NOTCH3 fusion much faster than mutant NOTCH3 (Figure 5B PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44964