Clearance of mutant NOTCH3 proteins.

<p>293 cells (n = 3 replicate wells per group) were transiently transfected with NOTCH3-luciferase constructs and split into identical 24 well chambers after 24 hours. After another 24 hours incubation, cells were treated with cycloheximide (100 ug/ml) in serum free media to arrest all protein synthesis, and groups of cells were analyzed at time points shown, when cell lysates were prepared and assayed for luciferase enzyme content. (A) The levels of NOTCH3-luciferase activity at the beginning of the time course did not differ significantly between any of the wildtype or mutant combinations. (B) Wildtype NOTCH3 ectodomain was more rapidly cleared than mutant NOTCH3 ectodomains. When cotransfections were performed with wildtype and mutant NOTCH3, proteins were cleared at an intermediate speed. (C) Quantification of a single time point (3 hours) is shown in a bar graph format to facilitate interpretation. The wild type protein is cleared most rapidly. Both CADASIL mutations slow clearance rate significantly. When mutants and wildtype protein were expressed together, there was an intermediate level of stability for R90C/wildtype protein. The C49Y/wildtype mixture was degraded at a similar rate as C49Y protein. Data is representative of three independent experiments. (*Significant compared to wildtype protein, #significant compared to all other groups; p<0.05.) Error bars represent standard deviations.