Figure 2.

Truncated SM associates with lipid droplets. HeLa cells were cotransfected with DsRed-ER and the indicated constructs for 24 h before treatment with 300 μM oleic acid for 16 h. A, immunoblotting was performed for N-terminal (HA)₃ tags, C-terminal V5 tags, and GAPDH. Dagger indicates a nonspecific band. B and C, cells were fixed, lipid droplets (LDs) were stained with BODIPY 493/503, and (B) anti-HA or (C) anti-V5 immunofluorescence was performed. Protein localization was determined by confocal microscopy. Scale bar indicates 10 μm for main images, and 1 μm for insets. White box denotes inset region. Images are representative. D, quantification of LD localization in (B) and (C). Data expressed as the mean intensity of HA or V5 staining on LD surface relative to the area surrounding LDs. Values >1 indicate protein-LD localization. LD localization is further defined in Experimental procedures and Fig. S2. Data presented as mean ± SD from n = 11 to 16 cells (∗∗∗∗p < 0.0001; ordinary one-way ANOVA). Images were collected across two independent experiments for each construct. SM, squalene monooxygenase; SM-N100, N-terminal regulatory domain of SM.