Fig. 7

Graf is essential for ligand-induced EGFR internalization in BG2-c2 neuronal cells. a Quantitative RT-PCR analysis showing Graf RNA expression in control and Graf-knockdown BG2-c2 cells. rp49 was a loading control. b, c Graf is required for Spi-induced internalization of EGFRs on the plasma membrane in BG2-c2 cells. Cells were transfected with a Flag-EGFR construct alone (control) or together with Graf dsRNA, and surface Flag-EGFR receptors were then prelabeled with anti-Flag antibody at 4 °C for 30 min. Cells were subsequently incubated with conditioned medium containing 0 or 10 ng/ml Spi-HA at 25 °C for 5 min for internalization of prelabeled cell surface Flag-EGFRs. After fixation of cells, surface (green) and internalized (red) Flag-EGFRs were sequentially labeled with secondary antibodies under nonpermeant and permeant conditions, respectively. b Single confocal sections through the middle of BG2-c2. Arrowheads indicate endosomal structures containing internalized EGFRs. c Quantification of internal-to-surface Flag-EGFR ratio in the control versus Graf-knockdown cells. Data are presented as means ± SEMs (n = 15 cells). Comparisons were made against Spi-treated control (***P < 0.001; one-way analysis of variance with Tukey–Kramer post hoc test). Scale bar, 5 μm