HGAL protein is myristoylated and palmitoylated and localizes in cellular membrane. (A) Amino acid sequences of the wild-type (WT), myristoylation and palmitoylation HGAL mutants. (B) HEK 293T cells transfected with plasmids encoding wild-type HGAL and G2A, C43A/C45A, and G2A/C43A/C45A myristoylation and palmitoylation mutants were used for metabolic labeling with [³H] myristic or [³H] palmitic acids. (C) Cellular distribution of HGAL in RCK8 (upper left image) and U2932 (upper right image) cell lines expressing WT HGAL-GFP (solid circle), G2A HGAL-GFP (solid square), C43A/C45A HGAL-GFP (open square), and G2A/C43A/C45A HGAL-GFP (open circle). HGAL distribution was determined from 3 random line profiles of a central stack of the confocal images of 12 to 15 cells. The edge of the cell was used to define the origin of the HGAL distribution and was determined by a sharp increase in the line profile. HGAL distributions were normalized to the total fluorescence from a line of 3 μm. Representative images are shown in the lower left and lower right images. Bar = 5 μm. (D) Expression of HGAL and its mutants was examined in cellular lysates using HGAL antibody and actin that was used as loading control.