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

Fig. 6.

Fig. 6

S-palmitoylation inhibits DGKε activity. HEK293 cells were transfected with indicated mDGKε-Myc variants or with empty vector (ev) with or without zDHHC17-HA and either homogenized by sonication after 48 h or subjected to cell lysis after 24 h in 1% NP-40 with or without a following mDGKε-Myc immunoprecipitation. The DGKε activity was determined in cell homogenates (A–C) or lysates and the mDGKε-Myc immunoprecipitates (D–F) using a fluorescence assay with mixed micelles of 1.45:2.03 mol% or 4.46:6.25 mol% NBD-SAG/SAG:PS, as indicated. A, D: Representative TLC separation revealing NBD-SAPA produced in homogenates of mDGKε-Myc-expressing cells (A) or in lysates of those cells and in the mDGKε-Myc-immunoprecipitates (D). (0), samples devoid of cell homogenate or lysate, in (0K) supplemented with the Myc-Trap Agarose. Reactions were carried out using 15 μg of total homogenate or lysate protein per sample or using mDGKε-Myc immunoprecipitate obtained from 75 μg of lysate protein. Lipids from 1/50 (A) or 1/10 (D) of the reaction mixture were separated by TLC. NBD-PDPA is used as a standard, it migrates more slowly on TLC than NBD-SAPA. B, E: Content of indicated overexpressed mDGKε-Myc variants (upper panel) and actin (lower panel) in cell homogenates, lysates, and mDGKε-Myc-immunoprecipitates revealed by immunoblotting with sheep anti-DGKε and mouse anti-actin antibody, respectively. GST-hDGKε is used as a standard. In (E) also zDHHC17-HA is revealed. Three micrograms of total homogenate protein (B) or 10 μg of the total lysate protein or one-third of the mDGKε-Myc immunoprecipitate (E) were applied per lane. C, F: Specific activity of indicated mDGKε-Myc variants calculated after subtraction of the activity of endogenous DGKs determined in control cells (ev). Molecular weight markers in kDa are shown on the right. Data are mean ± SD from eight (1.45 mol% SAG) or four (4.46 mol% SAG) experiments in (C) and from four experiments in (F). ∗ and ∗∗, significantly different at P < 0.05 and P < 0.01, respectively, from samples of WT mDGKε-Myc analyzed at corresponding mol% SAG. NBD-SAPA, 1-NBD-stearoyl-2-arachidonoyl-sn-glycero-3-phosphate.