Fig. 3.

FAM84B/LRATD2 is recruited to vesicle membranes in a GTP-dependent manner, interacts with AP1γ1 and Sec23A/B, and regulates ER-to-Golgi transport of EGFR but not ShhN or IGF2. (A) The vesicle formation assay was performed using the indicated reagents. The proteins in the vesicle fraction were analyzed by Western blot. (B) HEK293T cells expressing the FAM84B-HA were treated in 2 mM dithiobis(succinimidyl propionate) (DSP), and the cell lysates were incubated with beads conjugated with HA antibodies. After incubation, the bound proteins were analyzed by Western blot using the indicated antibodies. (C) HEK293T cells were transfected with control siRNA or siRNA against FAM84B/LRATD2. Day 3 after transfection, cells were lysed and analyzed by Western blot. (D, F, and H) HEK293T cells were transfected with control siRNA or siRNA against FAM84B/LRATD2. Twenty-four hours after transfection, cells were transfected with plasmids encoding the indicted constructs. On day 3 after knockdown, cells were incubated with biotin and cycloheximide for 15 min and the localization of the cargo proteins was analyzed by fluorescent microscope. (Scale bar, 10 μm.) (E, G, and I) Quantifications of the percentage of cells showing Golgi-localized cargo proteins in each experimental group (mean ± SD; n = 3; >100 cells counted for each experiment). ****P < 0.0001; *****P < 0.00001; n.s., not significant. Data shown in A–C are representative examples of three biological repeats.