Figure 8.

 Identification of a family of EH-containing proteins in mammals. (A) ³⁵S-Labeled lysates from NIH–3T3 (50 × 10⁶ TCA-precipitable counts) were incubated with the indicated GST fusion proteins displaying NPF-containing peptides (NPF–RAB, NPF–RAB-R, and NPF–NUMB, underlined in Fig. 1C), mutant peptides (NGR–RAB, DPF–RAB, and NPY–RAB, shown in Fig. 6A), or with GST. The first two lanes represent the same lysate immunoprecipitated with anti-eps15 antibody (EPS15) or with a control serum (PRE). Specifically bound proteins were fractionated by SDS–PAGE and visualized by autoradiography. The position of Eps15 is indicated. Molecular mass markers are indicated in kD. (B) Identification of four novel EH-containing mammalian sequences. The EST db was screened with the TBLASTN algorithm, using the three EH domains of Eps15 as a query. Only sequences displaying a P > 10⁻⁵ were considered for further analysis, and they are indicated with their database accession numbers. The alignment of these sequences to the second EH domain of Eps15 is shown, as obtained by a CLUSTAL4 algorithm modified by visual inspection. The W40735–W7520 sequence derives from a merging of two shorter EST sequences of the same gene.