to arrest protein synthesis. At defined time points beginning 24 hours after cycloheximide treatment, cells were analyzed for luciferase expression. Total firefly luciferase content was normal- ized to initial luciferase content (time 0). The decrease in luciferase activity over time was used to determine the effects of NOTCH3 mutations on clearance of NOTCH3 ectodomain. Notch Signaling Assays We used a cell co-culture system that was similar to previous studies [42,44]. For mRNA expression assays, we transfected A7R5 cells transfected (three wells per group) with NOTCH3 cDNA constructs with ligand expressing L cells expressing Jagged or Delta (or L cells without ligand as a control) [45]. After 24 hours, RNA from each well was prepared from cocultures, reverse transcribed, and quantified by real time PCR using beta-actin as a control. The average expression level for each group (n = 3 wells per co-culture; normalized to L cell controls) reflects target gene regulation by the Notch pathway. Finally, we determined the average levels of gene induction for three independent experi- ments. Primer sequences were designed to specifically measure A7R5 (rat) smooth muscle cDNA (and not mouse cDNA from ligand producing L cells). Sequences of these primers were: SM22:59-AAGAATGGCGTGATTCTGAGC-39 59-CTGCCT- TCAAGAATTGAGCC-39. SMA: 59- CAGACACCAGGGAG- TGATGGT -39 59CTTTTCCATGTCGTCCCAGT -39. Calpo- nin: 59-CAGTCAGCAGGGCATGACAG-39 59-AGTCATGCCA- GCCTGGCTG-39. SM-MHC: 59- CCGGCAACGCTACGAGAT - 39 59- AGAAGATTTTGCTCTGCCCA -39. For luciferase-based assays, H460 (that express only NOTCH3) or A7R5 cells (cells which express multiple Notch receptors) were transiently transfected with an expression clone (vector or NOTCH3 construct) in combination with the HES-luciferase reporter that reflects canonical NOTCH pathway activation [39,46], smooth muscle actin-luciferase (a 2.5 kb promoter construct), SM22-luciferase [47], or smooth muscle myosin heavy chain-luciferase mixed with a renilla luciferase standard. In some experiments, we applied recombinant, purified NOTCH3-Fc (1 ug/ml; 3 nM) or human IgG (control) protein to cocultures of reporter-transfected A7R5 cells (without co-transfection of NOTCH3 cDNA) and ligand expressing cells. To generate a Notch-insensitive promoter control, we mutated the RBP site at 2369 (from TTCCCAC to GATATCC; [48]) of the mouse SM22-luciferase reporter using PCR. The cells were allowed to recover overnight to enhance expression of the constructs. The following day, Notch was stimulated by overlaying Jagged1 or Delta1 expressing L cells (or parental L cells as a control) onto the Notch expressing monolayer [44,45]. After 18–24 hours, luciferase activity was determined from cell lysates; the ratio of firefly to renilla luciferase represented the activation of the Notch pathway in the transfected cell pool. Reporter studies were done in 48 well plates, and cells were transfected with 100 ng of reporter luciferase constructs, 1 ng of renilla luciferase control plasmid, and 100 ng of expression constructs (or vector control). Recombinant NOTCH3 Protein Purification Cell lines were prepared that stably expressed human NOTCH3 cDNA (corresponding 1,040 amino acids encoding the first 27 EGF-like repeats of NOTCH3; wild type or R90C mutant) fused to a myc tag and the Fc domain of murine IgG2 in pSec-Tag (Invitrogen). High expressing clones were selected by puromycin selection and expanded. After expansion, media was changed to OptiMEM (Invitrogen) overnight and this serum-free media was collected, frozen, pooled and passed over a protein A agarose column to affinity purify the recombinant NOTCH3 Cellular Effect of CADASIL Mutants of NOTCH3 fusion. Elution of the protein was performed at pH5.0, and the eluate was immediately neutralized with Tris pH 7.4 and dialyzed against PBS. The final protein solution was composed of only two bands, a major band (NOTCH3-Fc at approximately 150 kDa) and a minor band at 25 kDa (about 20% of the total mass by silver staining). Gel purification and mass spectroscopy of the minor band revealed it to be human IgG heavy chain made by 293 cell lines and which copurified with NOTCH3 proteins. To control for the IgG component of the protein preparation, all experiments compared the NOTCH3-Fc protein with purified human IgG as a control. Statistical Analysis All figures display means with standard deviations. Statistical analysis was performed using ANOVA with appropriate post hoc analysis and significance was assigned at p,0.05. Results We sought to answer four questions: 1) do wildtype or mutant NOTCH3 ectodomains physically interact with NOTCH1, 3, and 4 ectodomains; 2) do mutations in NOTCH3 affect detergent solubility; 3) do mutations in NOTCH3 impair protein clearance; and 4) does NOTCH3 overexpression affect Notch regulation of smooth muscle genes? NOTCH3 Homo- and Heteromeric Associations are Mediated by the Extracellular Domain Many NOTCH protein partners contain EGF-like repeats in their extracellular domains, including ligands Jagged, Delta, the Notch signal modifier thrombospondin-2 (THBS2; TSP2) [42], and LRP1 [49]; NOTCH1 has been shown to interact with itself [50,51] and several mutants of NOTCH3 homodimerize [36,38]. We therefore used immunoprecipitation assays to test whether NOTCH3 and additional mutants (the prototype R90C and C49Y) are capable of self-association through the EGF-repeat containing extracellular domain. 293A cells were cotransfected with two epitope tagged NOTCH3-extracellular domain con- structs (depicted in Figure 1). In Figure 2A, we demonstrate that wildtype NOTCH3 ectodomain tagged with HA is capable of interacting with WT and mutant NOTCH3 ectodomains, as assessed by coprecipitation of WT-HA protein with all three myc- tagged NOTCH3 ectodomains. Negative control transfections (e.g. WT-myc protein alone or WT-HA protein alone) did not yield coprecipitated proteins with the converse tag. Mixing of NOTCH3 proteins expressed separately did not result in coprecipitated protein, indicating that interactions are favored by co-expression of proteins within the same cell (not shown). Similarly, in Figures 2B and 2C, we show that precipitation of mutant NOTCH3 proteins R90C and C49Y pulls down both wildtype and mutant proteins. Quantification of band intensities of all immunoprecipitation experiments (repeated over 8 times) demonstrated that there was no significant difference between the coimmunoprecipitations of any NOTCH3 combination. Under identical assay conditions, several additional proteins do not coprecipitate with the NOTCH3 ectodomain (see Figure S1 for an example). Multiple EGF-like repeats in NOTCH are predicted to bind calcium [52]. Moreover, the addition of EDTA to cells expressing NOTCH proteins has been reported to stimulate proteolysis and release of the NOTCH3 extracellular domain, suggesting a conformational change resulting from calcium chelation [52]. However, immunoprecipitations prepared and washed in buffers containing either calcium or magnesium showed no difference in PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44964