HGAL myristoylation and palmitoylation modifications mediate its localization to membrane lipid rafts. Detergent-free fractionation by sucrose-density centrifugation was done on RCK8 cells (A) and U2932 cells (B) stably transfected with plasmids encoding V5-tag WT HGAL, HGAL myristoylation (G2A), HGAL palmitoylation (C43A/C45A), and HGAL palmitoylation and myristoylation (G2A/C43A/C45A) mutants. Individual fractions were immunoblotted with the indicated antibodies. Input represents 10% of the sample taken from cellular lysate after sonication and before centrifugation. Lyn, transferrin receptor, and BLNK were used as lipid raft, bulk membrane, and cytoplasm markers, respectively. HGAL was detected using V5-tag antibody. Results are representative of 3 independent experiments. (C) Distribution of HGAL in RCK8 and U2932 cells transfected with WT HGAL and HGAL mutants was generated by analyzing total densitometry in lipid raft fractions (2-5) and bulk membrane/cytoplasm fractions (6-10) using ImageJ and Origin 7 software. Data are expressed as the mean ± standard error of the mean of triplicates. P values are shown for each cell line.