Fig. 2: ARF6 is required for the sorting of EGFR to PM.
From: Targeting EGFR-dependent tumors by disrupting an ARF6-mediated sorting system
a A schematic illustration of the retention using a selective hook (RUSH) system to study the effect of ARF6 on sorting EGFR from Golgi to PM. b On day 0, HeLa cells were transfected with Streptavidin-Golgin-84 and WT or 3CS of EGFR-HA-SBP (0.5 μg each). On day 1, cells were fixed at 0 or 1 h after 100 μM d-biotin treatment and immunostained using anti-HA and anti-GM130 antibodies. c On day 0, WT or 9CS EGFR-HA-SBP was transfected with Streptavidin-Golgin-84 into HEK293T. On day 1, cells were washed with PBS and crosslinked with 2 mM DSP on ice for 2 h, which was stopped by 0.1 M Tris (pH 8.0). Immunoprecipitations were performed using anti-HA beads. The pellets were subjected to silver staining and mass spectrometry. d Twenty-four hours after transfection with EGFR-HA-SBP and Streptavidin-Golgin-84, WT and ARF6−/− HeLa cells were treated with 100 μM d-biotin and immunostained as in (b). e–g ARF6−/− HeLa cells were transfected with Streptavidin-Golgin-84 (e–g), WT (e, g) or 3CS (f) of EGFR-HA-SBP and WT (e, f) or G2A (g) of ARF6-Flag (0.5 μg each). Cells were treated with 100 μM biotin and immunostained as in (d). Fluorescent intensity was quantified using ZEN2.3. Arrows indicate the quantification area. h, i HeLa cells were transfected with indicated plasmids with Streptavidin-Golgin-84. Proximity ligation assay was performed following the manufacturer’s instructions. Intensities were quantified and plotted (i). Data were presented as mean ± SEM, EGFR-HA-SBP, and ARF6-Flag, n = 28; 3CS-HA-SBP and G2A-Flag, n = 24. A two-sided Student’s t-test was performed. j, k HEK293T cells were transfected with indicated plasmids. Eight hours later, cells were incubated with 1 μM nocodazole overnight with or without 100 μM 2-BP, washed twice with PBS, and incubated in a fresh medium for another 0.5 h. Cells were treated with 2 mM DSP as in (c). Cell lysates were subjected to immunoprecipitation with anti-Flag M2 beads. Cells were pretreated with 2-BP for 24 h before harvest (j). Scale bars were as indicated. Source data are provided as a Source Data file.
