Skip to main content
. 2023 Nov 24;65(1):100480. doi: 10.1016/j.jlr.2023.100480

Fig. 1.

Fig. 1

DGKε is S-palmitoylated at the cysteine located at the cytosolic end of its N-terminal transmembrane fragment. A: The amino acid sequence of the N-terminal fragment of hDGKε and mDGKε. Amino acids mutated in this study are indicated by red font. The predicted transmembrane fragment is indicated by the solid line. The complete mDGKε/hDGKε consists of 564/567 amino acids. B: Scheme of the click chemistry procedure. C, D, F–H: HEK293 cells were transfected with plasmid encoding WT mDGKε-Myc (C, D, F, G) or hDGKε-Myc (H) or their indicated mutant forms. C, D: After 48 h, cells were subjected to metabolic labeling with 50 μM 17ODYA or exposed to 0.05% DMSO carrier as control (−17ODYA) for 4 h and lysed. mDGKε-Myc was immunoprecipitated with anti-Myc alpaca antibody and subjected to click chemistry reaction with IRDye 800CW-azide. C, upper panel: In-gel fluorescence showing mDGKε-Myc labeling with 17ODYA followed by IRDye-azide. C, lower panels: Efficiency of immunoprecipitation determined by immunoblotting with mouse anti-Myc antibody. The content of mDGKε-Myc and actin in input lysates is shown on the right. Arrowhead indicates a band recognized unspecifically by the anti-Myc antibody. D: The extent of mDGKε-Myc palmitoylation. mDGKε-Myc fluorescence was determined by densitometry and normalized against the content of respective Myc-tagged DGKε variant in immunoprecipitates. E: Scheme of the ABE procedure. F–H: After 48 h of transfection, cells were lysed and proteins were subjected to the ABE procedure involving treatment with HXA (HXA+) or not (HXA−), biotinylation, and capture of originally S-palmitoylated proteins on streptavidin-agarose beads. F, H, upper panels: S-palmitoylated mDGKε-Myc (F) and hDGKε-Myc (H) eluted from the streptavidin-agarose beads revealed with mouse anti-Myc antibody. F, H, lower panels: Transferrin receptor (Tfr), an S-acylated protein, eluted from the beads. The content of total mDGKε-Myc, hDGKε-Myc, and TfR in input lysates is shown on the right. G: The extent of mDGKε-Myc S-palmitoylation. The content of mDGKε-Myc in eluates and input lysates was determined by densitometry, normalized against TfR, and the ratio of S-palmitoylated mDGKε-Myc (eluates) to total mDGKε-Myc (lysates) is shown. Molecular weight markers in kDa are shown on the right. Data shown are mean ± SD from four experiments. ∗, ∗∗∗, Significantly different at P = 0.05 and P < 0.001, respectively, from cells expressing WT DGKε. ev, empty vector.