Figure 3.

The first two of five Rabenosyn-5 NPF motifs are critical for binding to EHD1. (A) Bacterially expressed, recombinant GST-EHD1 was incubated with full-length wild-type Rabenosyn-5 (FL), or various ³⁵S-labeled Rabenosyn-5 transcription/translation products with deletions and/or truncations, and the bound proteins were analyzed by SDS-PAGE and autoradiography. Five percent of the total input is shown in the top panel, and the bound Rabenosyn-5 products are shown in the bottom panel. (B) Individual NPF motifs were mutated from NPF to APA (ΔNPF1–5), or the first two NPF motifs were mutated to APA (ΔNPF1,2). ³⁵S-labeled Rabenosyn-5 transcription/translation products of wild-type Rabenosyn-5 or NPF mutants were pulled down with GST-EHD1. Five percent of input is shown in the top panel, whereas bound Rabenosyn-5 products are shown in the bottom panel. (C) Densitometric analysis of the binding experiment depicted in B is shown in histogram format.