Figure 3.
The A683T mutation affects S1 cleavage of the NOTCH1 receptor. (A) Western blot analysis of N-terminally HA-tagged NOTCH1 protein. NIH3T3 cells transfected with Notch1 expression vectors were lysed and the expression of NOTCH1 protein was assessed with an anti-HA antibody. HA-NOTCH1 bands are observed at 300 and 180 kDa, representing full-length (p300) and S1 cleaved (p180) NOTCH1 receptor. All proteins are expressed robustly, and to similar levels as seen in wild-type. (B) The levels of p300 and p180 Notch1 proteins were quantitated in western blots, demonstrating that the NOTCH1A683T variant undergoes S1 cleavage at reduced levels. Results are expressed as the percent of total protein found in the p180 band. Error bars represent the standard deviation from the mean of at least four experiments. ** indicates P < 0.01 by ANOVA followed by Dunnett’s post hoc.