Fig. 8.

Hyperphosphorylation causes disassociation of Numb complexes: A, Stable cell lines expressing either 3XFLAG Numb p66 or p72 were treated for 20 min with either 50 nm Calyculin A or equivalent volume DMSO (Vehicle). Western blot shows a band shift due to hyper-phosphorylation of both Numb isoforms when treated with Calyculin A. An anti α-tubulin probe was used to indicate equal protein loading. B, SRM-MS was performed on FLAG immunoprecipitates from cell lines expressing Numb p66 or p72 to analyze effect of phosphorylation (Calyculin A treatment) on Numb interacting proteins. Relative amounts of each interacting protein are graphed as a Calyculin A-induced fold change compared with DMSO-treated controls, and normalized to the amount of Numb protein in each sample. Error bars represent standard error. Asterisks represent statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001).