Fig. 2. (A) Western blotting of sucrose density-gradient fractions from HepG2. An equal volume from each fraction was loaded. Both ADRP and Rab18 were strongly present in the top floating LD fraction (#1). There was also some Rab18 in the bottom fractions containing soluble and membrane proteins (#7, #8). EEA1, Lamp1, syntaxin 6, and calnexin were detected only in the bottom fractions (#6-8). (B) Double labeling of endogenous Rab18 (red) and LDs (green) in HepG2 cells. LDs were stained with BODIPY 493/503. Antibody labeling for Rab18 was concentrated around LDs. Note that there were LDs that were not associated with anti-Rab18 (arrowheads). Scale bar: 10 µm. (C) Double labeling of endogenous Rab18 (green) and endosomal markers (red). EEA1 and transferrin receptor (Tf-R) (upper panel), and lysobisphosphatidic acid (LBPA) and Lamp1 (lower panel) were used as markers for early and late endosomes, respectively. Rab18 did not show co-distribution with any of the endosomal markers. Scale bars: 10 µm.