Fig. 2

EGFR was identified as a novel KDELR-interacting protein. A Volcano plot representing results of KDELR BioID. HeLa cells were transfected with KDELR-BirA^* or KDELR-myc for 24 h and incubated with 50 μM biotin for 6 h, prior to cell disruption. Proteins in close proximity to KDELR- BirA^* were enriched by streptavidin beads and subjected to mass spec analysis. The logarithmic ratios of protein intensities identified in KDELR-BirA^* versus control (KDELR-myc) were plotted against negative logarithmic p-value of the t test performed from triplicate experiments. KDELR, surface receptor and clathrin-related proteins are marked in red. B Volcano plot showing results of surface-KDELR pulldown-mass spec experiment. Halo-KDELR expressing on the cell surface of HeLa cells were labeled with non-membrane permeable biotin-conjugated Halo ligand. Proteins in cell lysates were precipitated by streptavidin beads and analyzed by mass spec. The logarithmic ratios of protein intensities in HeLa cells with biotin-conjugated Halo ligand incubation versus DMSO were plotted against negative logarithmic p-value of the t test performed from triplicate experiments. C Co-IP experiments in HeLa cells overexpressing mCherry or KDELR-mCherry using anti-RFP beads confirmed that EGFR and TfR as KDELR-interacting proteins. D, E The interaction between EGFR and KDELR brought two halves of Venus protein together to generate Venus signals. HeLa cells transfected with Halo-KDELR-Flag-VN and EGFR-myc-VC, HGFR-myc-VC, or TfR-myc-VC were treated with 50 μM TAEKDEL peptide or DMSO at 4 °C for 30 min, followed by incubation at 37 °C for 30 min. Cells were then fixed, stained with indicated antibodies, and observed under confocal microscope (D). Venus signal ratio of TAEKDEL- versus DMSO-treated was statistically analyzed by one-way ANOVA with Tukey’s multiple comparisons (E). n = 20 cells pooled from 3 independent experiments. ****: P < 0.0001. Scale bar = 10 μm