Fig. 1. Localization of endogenous ARFRP1. (A) HeLa cells were fixed and double-stained for ARFRP1 and either the AP-1 -adaptin subunit, GGA3, golgin-245 or GM130 followed by Cy3-conjugated anti-mouse IgG and Alexa Fluor 488-conjugated anti-rabbit IgG. Bar, 20 µm. (B) Immunoblot analysis for ARFRP1 in mock transfected HEK293 and HeLa cells, and HeLa cells transfected with HA-tagged ARFRP1. An asterisk indicates the positions of endogenous ARFRP1 and a double asterisk indicates exogenously expressed ARFRP1-HA. (C) Cryo-thin sections of HeLa cells expressing GFP-CI-MPR were double-labeled with mouse anti-GFP and rabbit anti-ARFRP1 (arrows) antibodies, which were then detected by two secondary antibodies conjugated with 15 nm- and 10 nm-colloidal gold particles, respectively. Arrowheads indicate two clathrin-coated vesicles, one of which contains a GFP-CI-MPR signal. Go, Golgi stack. Bar, 0.1 µm.