Figure 1.
A Minimal EPS15 Interaction Motif
(A and B) GST pull-down assays with rat brain cytosol and 250 μg of GST and either 50 or 250 μg of the indicated GST-EPS15 fusion proteins. SDS-PAGE-separated supernatant (S, 2%) and pellet (P, 10%) fractions were stained or immunoblotted with antibodies against Fcho2.
(C) Location of DPF triplets in the EPS15 C-terminal domain, with the relative location of the various fragments tested below. Alignment of Homo sapiens (Hs) EPS15, EPS15R, and DAB2 sequences corresponding to the minimal motif, boxed with red for identity and pink for similarity.
(D) GST pull-down assay with purified FCHO1 μHD and ∼50 μg of either GST or GST-μHD fusion protein in the presence of carrier BSA. SDS-PAGE-separated supernatant and pellet fractions were stained or immunoblotted with an anti-FCHO1 monoclonal antibody.
(E) Representative ITC experiments with the Danio rerio (Dr) Fcho1 μHD and EPS15 616–638 peptide or the Hs FCHO1 μHD titrated with the 616–638 or indicated EPS15 peptides. KD, reaction stoichiometry (n), and errors were calculated from a minimum of four runs.
(F) Cartoon representation of GST-EPS15-FCHO1 μHD chimeras, with the specific EPS15 sequence for each variant preceding the GlyProSer spacer shown below.
(G) GST pull-down assay using HeLa cell lysate and 250 μg of either GST or GST-μHD fusion proteins as indicated. Separated supernatant and pellet fractions were stained or replicates immunoblotted with the indicated antibodies as in (A).