Fig. 4

EGFR mediates the endocytosis of KDELR induced by KDEL ligand. A-C HeLa cells co-transfected with Halo-KDELR and mCherry-clathrin were stained with non-membrane permeable HaloTag Alexa Fluor 488 ligand and treated with 50 μM TAEKDEL peptide (A) or DMSO (B) at 4°C for 30 min, prior to incubation at 37°C for 0, 15, 30 min. Cells were stained with anti-EGFR antibody and observed by Zeiss LSM880 microscope. Regions of interest highlighted by dashed white lines were magnified. Scale bar = 10 μm. The co-localization of EGFR and KDELR in the Golgi area was represented by Pearson’s coefficient and analyzed using two-way ANOVA with a Sidak’s test (C). n = 20 cells pooled from 3 independent experiments. D, E The endocytosis of KDELR was impaired in EGFR-depleted cells. HeLa cells depleted of EGFR were transfected with Halo-KDELR and mCherry or EGFR-mCherry for 18 h and stained with non-membrane permeable HaloTag Alexa Fluor 488 ligand for surface-expressed KDELR. Cells were then treated with 50 μM TAEKDEL peptide at 4 °C for 30 min, prior to incubation at 37 °C for 0 or 30 min. Endogenous clathrin was stained with antibody, followed by confocal image analysis (D). The percentage of the surface green fluorescence signals in the total green signals was quantified and analyzed by two-way ANOVA with Sidak’s test (E). n = 20 cells pooled from 3 independent experiments. ****: P < 0.0001. Scale bars = 10 μm. F KDEL ligand did not induce the endocytosis of KDELR-ΔCT. HeLa cells depleted of KDELR were transfected with Halo-KDELR or Halo-KDELR-ΔCT and treated with 50 μM TAEKDEL peptide at 4 °C for 30 min. Cells were then incubated at 37 °C for 0 or 30 min, prior to labeling by sulfo-NHS-LC-Biotin at 4 °C for 10 min. Total surface proteins were pulled down by streptavidin beads and analyzed by immunoblotting. Relative protein levels on the cell surface were subjected to two-tailed, unpaired t tests (n = 3 independent experiments). *: P < 0.05. G TAEKDEL ligand incubation improved the binding between KDELR and EGFR. HT1080 stably overexpressing KDELR-mCherry cells were treated with 50 μM TAEKDEL peptide or DMSO at 4 °C for 30 min, prior to incubation at 37 °C for 30 min. Cell lysates were then prepared and subjected to RFP-IP analysis