Fig. 4: Lys3 of ARF6 recruits the exocyst complex to facilitate EGFR budding from Golgi.

a On day 0, HEK293T cells were set up at 7 × 10⁵ cells per 10-cm dish. On day 2, cells were transfected with EGFR-Flag/pCDH-puro and ARF6-HA/pCDH-puro (4 μg each). On day 3, cells were treated and subjected to tandem affinity purification using anti-HA and anti-Flag conjugated beads as described in Methods. The eluted fraction was analyzed by mass spectrometry. b On day 0, HeLa cells were set up as in Fig. 1c. On day 1, cells were infected with lentivirus expressing shRNAs of EXOC2, EXOC5, or EXOC6 on a pLL3.7 vector. On day 3, cells were transfected with EGFR-Flag/pCDH-puro. On day 4, cells were harvested for immunostaining. Scale bar, 10 μm. c HEK293T cells were set up and transfected with ARF6-HA/pCDH-puro and each of the Flag-tagged subunits of the Exocyst complex on a pcDNA3.3 vector. On day 3, cells were harvested for immunoprecipitation using anti-Flag M2 beads. The input and pellet fractions were subjected to western blot using indicated antibodies. d HEK293T cells were co-transfected with Flag-EXOC2 and HA-tagged ARF6, ARF5, N6C5, or N5C6, harvested for immunoprecipitation with anti-Flag M2 beads and subjected to western blot. The input and pellet fractions were subjected to western blot using indicated antibodies. e HEK293T cells were set up, co-transfected with Flag-EXOC2/pCDH-puro and N-terminal mutants of ARF6, and subjected to immunoprecipitation as in (c). The input and pellet fractions were subjected to western blot using indicated antibodies. f WT and mutants of ARF6-Flag were expressed and purified from HEK293T cells. GST and GST-EXOC2 were bacterially expressed and purified. The proteins with indicated combinations were incubated and subjected to GST-pull down analysis as described in Methods. The input was subjected to Coomassie staining, and the pellet fractions were subjected to western blot using the indicated antibodies. Source data are provided as a Source Data file.