Fig. 5

TAEKDEL peptide greatly increases KDELR oligomerization, but only moderately enhances EGFR oligomerization. A KDEL ligand induced moderate increase of EGFR oligomerization. HeLa cells co-expressing EGFR-myc with mCherry (negative control) or EGFR-mCherry were incubated with 30 nM EGF or 50 μM TAEKDEL peptide at 4 °C for 30 min. Cells were then processed for IP with mCherry and western blotting. B-G HeLa cells co-transfected with EGFR-myc-VC/EGFR-Flag-VN (B-D) or Halo-KDELR-V5-VN/Halo-KDELR-myc-VC (E–G) were treated with 50 μM TAEKDEL peptide or 30 nM EGF at 4 °C for 30 min, followed by incubation at 37 °C for 0, or 30 min. Cells were then fixed, stained with indicated antibodies, and observed under confocal microscope. Venus signal intensity ratio of 30-min incubation at 37°C versus 0-min was quantified using two-tailed t-test (D for results of B and C, G for results of E and F). n = 20 cells pooled from 3 independent experiments. *: P < 0.05. **: P < 0.01. Scale bars = 10 μm