|
Fig. 6
The subcellular localization of Cers2 protein is altered by increased sphingosine levels.
HEK293T cells transfected with a Cers2-GFP expression plasmid were examined after 6 hr exposure to 10 uM sphingosine in culture. (A) Cells treated with sphingosine (Sph) show subcellular relocalization of Cers2b-GFP (green) with more overlap within nuclear regions (DAPI, blue) as seen in the z-stack (side panels). (B–C) Western blotting analysis of lysates probed with antibodies specific to Cers2, GAPDH (as a control for cytoplasmic proteins), TBP (as a control for nuclear proteins) or LaminB1 (as a control for nuclear membrane associated proteins). (B) Subcellular fractionation into cytoplasmic (Cyto) and nuclear (Nuc) lysates confirms increased nuclear association of Cers2 in cells treated with sphingosine. (C) Fractionation using a nuclear membrane isolation kit indicates Cers2 is most enriched in the nuclear membrane fraction.