Cellular Effect of CADASIL Mutants of NOTCH3 ectodomains and strongly inhibited Notch function. Since NOTCH regulates crucial smooth muscle genes, we tested whether the overexpression of NOTCH3 affects the regulation of genes known to regulate smooth muscle homeostasis. protein. A7R5 cells were transfected in 48 well plates with 200 ng of total DNA and 1 ul of Lipofectamine 2000 per well. Transfection efficiencies typically ranged from 25–50% when assessed using GFP expression plasmids. Materials and Methods Materials Cell culture products were obtained from Invitrogen (Carlsbad, CA) unless noted; puromycin was purchased from Alexis (San Diego, CA). All other chemicals were obtained from Sigma (St. Louis, MO) unless indicated otherwise. Construction of cDNA Clones Encoding Human NOTCH3 Please see Figure 1 for representation of constructs. We constructed a clone for NOTCH3 in the expression vector pCMV-Sport6 (Invitrogen, Carlsbad, CA) by using publicly available cDNA clones and RT-PCR from human brain RNA [39]. We created two mutants of NOTCH3 (R90C and C49Y; mutation sites represented by black bars) by PCR based mutagenesis [40]. Epitope tagged ectodomain fragments were created by insertion of oligonucleotides encoding the appropriate tags into the NotI site of the human NOTCH3 cDNA, resulting in an open reading frame that corresponds to the first 33 EGF-like repeats of the molecule. NOTCH3-luciferase fusions were created by splicing luciferase from pGL3 (Promega) into the same internal Not1 site and a 39 XbaI site just beyond the end of the coding sequence. The 59 fragment of full length mouse NOTCH4 cDNA (a gift of Jan Kitajewski) spanning base pairs 1–4309 was cloned in frame between the EcoRI and NotI restriction sites of the pcDNA4V5zeo vector (Invitrogen) to obtain a C-terminal V5- tagged NOTCH4 extracellular domain. Cell Culture 293A (Qbiogene, Irvine, CA), A7R5 rat aortic smooth muscle, and H460 human lung cancer cells were grown in DMEM supplemented with 10% FBS. 293A cell lines were generated by cotransfection using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. DNA for transfec- tions included a 1:20 mixture of a puromycin resistance plasmid (Ambion, Austin, TX) and full length human NOTCH3 clones (wildtype and R90C or C49Y mutants). Lines containing empty vector were prepared as controls. Clones were isolated after two weeks of selection in puromycin (2 ug/ml). Experiments using cell lines were verified using at least one additional independent clone, and all NOTCH3 clones selected expressed equivalent amounts of Immunoblotting Western blots were performed using standard methods onto PVDF membranes using commercial antibodies for NOTCH3, HA (F7), and myc (9E10) (R&D and Santa Cruz Biotechnology; 1:2000 dilution). Detection was performed using enhanced chemiluminescence (Amersham, Pisctaway, NJ) or by using infrared conjugates of secondary antibodies (Rockland) detected by a LiCor flatbed Odyssey imager. Immunoprecipitation Plasmids encoding the extracellular domain of human NOTCH1 or NOTCH4 (tagged with V5 [41]) and/or NOTCH3 tagged with HA or myc epitopes were transfected into 293A cells. Cell lysates were prepared in modified RIPA buffer, sonicated, and cleared by centrifugation [42]. Supernatants were incubated with antibodies (1 ug) and then mixed with protein G agarose beads overnight. Beads were pelleted and washed with modified RIPA buffer three times. Detergent Extraction of Cell Lysates Experiments were performed based on a protocol developed by Wang et al. [43]. Cell lines were plated at desired density and approximately 250 ug of protein was analyzed as follows: cells were rinsed with PBS, scraped, lysed by sonication in AO buffer (Tris 10 mM, pH 7.4, 100 mM NaCl, protease cocktail (Pierce, Rockford, IL) and sedimented at 15,0006G in an Eppendorf microfuge for 30 minutes at 4C. The supernatant was designated S1. The pellet was sonicated in A1 buffer (AO with 0.5% Triton) and pelleted as before. The supernatant was designated S2. The pellet was sonicated in A2 buffer (AO with 1% Triton and 0.5% deoxycholic acid) and recentrifuged; the supernatant was desig- nated S3, and the pellet (S4) was resuspended in sample buffer. Fractions S1 through S4 were all resuspended in sample buffer and analyzed by Western blotting for NOTCH3 content. Degradation Assays We determined the kinetics of NOTCH3 ectodomain clearance from cells by genetically fusing the NOTCH3 ectodomain to firefly luciferase. 293A cells were transfected with NOTCH3- luciferase fusions (see Figure 1) for 24 hours in 24 well plates. Cells were treated with 100 ug/ml cycloheximide and serum free media Figure 1. Constructs used in this study. Schematic representation of constructs used in this study, as described in the methods. The positions of the mutations examined in the present study are indicated by small dark rectangles. Wildtype and mutant constructs were all cloned into pCMV- Sport6 (Invitrogen). Tm = transmembrane domain of NOTCH3, which is deleted in tagged constructs. The constructs were prepared in pCMV-Sport6 (Invitrogen). Tagged NOTCH3 ectodomain constructs were prepared by replacing the C-terminal 3 kb of NOTCH3 with either an HA or myc tag or with the full coding sequence of firefly luciferase. The HA and myc tagged proteins run on SDS gels at an apparent molecular weight of 175 kDa. doi:10.1371/journal.pone.0044964.g001 PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44964