Fig. 3

KDELR likely interacts with EGFR through its C-tail. A Schematic illustration of split-ubiquitin membrane yeast two hybrid (MYTH) assay. KDELR was fused to Cub and a transcription factor in the bait vector. ACBD3, EGFR, GLUT4, and ITGA5 were subcloned into the Nub-containing prey vector. Interaction between KDELR and a prey protein combines Cub and Nub to release the transcription factor and allow the transcription of HIS3, ADE3, and LacZ genes. B KDELR interacted with ACBD3 and EGFR, but not with GLUT4 and ITGA5, in MYTH assay. Yeast cells were transformed with indicated bait and prey plasmids and grown on transformation selection plates depleted of tryptophan and leucine (DDO, upper panel) or on interaction selection plates depleted of tryptophan, leucine, histidine, and adenine, plus 40 μg/ml X-α-Gal (QDO, bottom panel). C Schematic representation of EGFR chimera with EGFR transmembrane domain swapped for HGFR transmembrane motif. D EGFR chimera co-immunoprecipitated with KDELR-Flag. HeLa cells overexpressing KDELR-Flag and EGFR-mCherry or EGFR-(HGFR-TM)-mCherry were lysed and subjected to IP with anti-Flag sepharose, followed by immunoblotting analysis. E KDELR depleted of C-tail did not interact with EGFR. HeLa cells transfected with mCherry, KDELR-mCherry, or KDELR-ΔCT were processed for mCherry IP and western blotting. F The C-terminal tail of KDELR pulls down EGFR. Recombinant GST and GST-CT (KDELR-CT) proteins conjugated on glutathione beads were incubated with cell lysates prepared from A431 cells and subjected for immunoblotting with indicated antibodies