Fig. 7.

EPS15 preferentially associates with p72: A, HEK293T cells were transfected with FLAG Numb p72, p66 or empty vector, and lysed in Nonidet P-40 lysis buffer. Immunopurifications with either anti-FLAG antibody or normal Mouse IgG (control IP) was performed and isolated protein complexes were separated by SDS-PAGE and analyzed by immunoblot using antibodies to EPS15 and to FLAG. Immunoblot shown is blot is representative of three replicate experiments. Western immunoblot chemiluminescence signals for EPS15 were quantified and normalized to amount of Numb in each sample. This analysis showed that the mean ratio of EPS15 to p72 Numb (0.23 se = 0.07) was significantly greater than the ratio of EPS15 to p66 Numb (0.19 se = 0.06) (p = 0.027). B, Representative example of immunofluorescence images of Hela cells transiently transfected with either 3XFLAG Numb p66, or p72 and subjected to a proximity ligation assay (PLA) using antibodies against FLAG and endogenous EPS15. To confirm Numb expression, cells were stained with anti-Numb (green), and Numb signal in overexpressing cells is noticeably greater than untransfected cells and very few PLA events are observed in untransfected cells. C, PLA events (red) in greater than 50 cells from a minimum of three biological replicates were quantified. Average counts per cell were calculated and graphed (Average PLA events: Numb/Eps15 PLA: p66 237.3 ± 8.3, p72 307.0 ± 24.5; Numb/TfR p66 234.0 ± 14.6, p72 249.9 ± 29.7; TfR/Eps15 p66 150.9 ± 12.0, p72 149.8 ± 19.0). Error bars represent standard error of the mean; double asterisk indicates statistical significance, p < 0.05. (Numb/Eps15 p66 n = 161, p72 n = 183, Numb/TfR p66 n = 122, p72 n = 105, TfR/Eps15 p66 n = 80, p72 n = 74).