WT ELOVL4 and ELOVL4 SCA34 mutants L168F and W246G localize in the ER. A: ARPE-19 cells transduced with MYC-ELOVL4 constructs were immunostained for MYC (red) and CALNEXIN (green). WT, L168F, and W246G colocalized with ER transmembrane marker Calnexin. (Scale bar, 10 μm). B: Localization of WT ELOVL4 and SCA34 L168F and W246G mutant proteins by subcellular fractionation. Equal amounts of protein from membrane, cytoplasmic, and nuclear fractions of ARPE-19 cells transduced with MYC-ELOVL4 constructs were subjected to Western blot analysis using MYC antibody. CALNEXIN was used as a marker for the membrane (M), MEK1/2 as a loading control for the cytoplasmic fraction (C), and Histone 3 for the nuclear fraction (N). ELOVL, elongation of very long-chain fatty acid; SCA, spinocerebrellar ataxia.