Figure 3

GGA2 VHS-GAT recognizes EGFR juxtamembrane (jxt) region. (a) Domain structures of EGFR and GGA2 mutants used in the binding assay are shown. Numbers indicate amino acid sequences of the proteins. N108A mutation site is indicated in VHS-GAT. TMD, transmembrane domain. (b and c) HEK293 cells were transfected with GFP tagged with entire cytoplasmic domain of EGFR (cyto), or with its jxt, kinase, or tail region. Cell lysates were mixed with glutathione sepharose and GST-GGA2 VHS-GAT (GST-VG[WT]) or its N108A mutant (GST-VG[NA]). Bound proteins were then eluted and analyzed using SDS-PAGE and immunoblotting with anti-GFP or -CIMPR (only for [b]) antibody as indicated (Ab). (d) Cell lysates from HEK293 cells expressing GFP-jxt were pulled down using GST fusion proteins containing wild type (WT) or N108A-, N211A-, F280R-, R281E, or L293A-mutated forms of GGA2 VHS-GAT, or with GST proteins fused with GAT, GAE, or the VHS region of GGA2. Bound proteins were immunoblotted using indicated antibodies (Ab). Ponceau stainings are shown at the bottom. Arrows and arrowheads indicate GST and GST-VHS-GAT, respectively. Uncropped blots are presented in Supplementary Fig. 6.