Fig. 3: The N-terminus of ARF6 is required for EGFR budding from Golgi.

a On day 0, ARF6^−/− HeLa cells were set up and transfected with EGFR-Flag/ pCDH-puro and HA-tagged ARF6, ARF5, N5C6, or N6C5 on pCDH-puro (0.5 μg each). On day 2, cells were serum-starved overnight. On day 3, cells were fixed and subjected to immunostaining using anti-HA, anti-Flag, and anti-GM130 antibodies. Quantification of the fluorescent intensity was performed using ZEN2.3. The domain structures of the ARF proteins were illustrated in the left panel. Arrows indicate the area that was used for quantification. b ARF6^−/− HeLa cells were set up, transfected, and subjected to immunostaining as in (a), except that different mutants of ARF6-HA/pCDH-puro were used. On day 3, cells were harvested and subjected to immunostaining using anti-HA, anti-Flag, and anti-GM130 antibodies. c WT HeLa cells were set up and transfected with EGFR-Flag/pCDH-puro and WT or mutants of the N-terminus of ARF6 fused with GFP (N6-GFP)/pCDH-puro. On day 3, cells were harvested and subjected to immunostaining using an anti-Flag antibody. Scale bar, 10 μm. d HEK293T cells were set up and transfected with indicated plasmids. On day 2 (8 h after transfection), cells were treated with 1 μM nocodazole overnight. On day 3, after the removal of nocodazole for 0.5 h, cells were treated with 2 mM DSP in ice-cold PBS for 2 h. The crosslink was stopped by Tris (pH 8.0, 0.1 M) at 4 ^°C for 15 min. Cells were lysed and subjected to immunoprecipitation using an anti-Flag antibody. Input and pellet fractions were subjected to western blot using indicated antibodies. Scale bars were as indicated. Source data are provided as a Source Data file.