Fig. 3.

The EH domain of REPS1 mediates binding to Numb. A, Schematic diagram of Numb indicating location of mutant constructs used to map the REPS1 binding site and schematic diagrams of REPS1 and EPS15. B, GST-fusion proteins of the isolated EH domain of REPS1 or EH2 domain of EPS15 were purified and immobilized on glutathione agarose and incubated with HEK293T cell lysate. The bound complexes were washed, separated by SDS-PAGE and immunoblotted with antibodies directed against Numb. C, HEK293T cells were transfected with HA-tagged Numb p66, p72 or carboxy-terminal truncation mutants (HA-p66ΔC, HA-p72ΔC), lysed, and mixed with immobilized GST fused to the EH domain of REPS1 or the EH2 domain of EPS15. Complexes were washed and analyzed by immunoblot with anti-HA antibodies.