Figure 4

ω 3PUFAs increase the ubiquitination of HMGCoAR by activating the E3 ligase Trc8. (A) The microsomal fractions from HT29 and HT29-dx cells were incubated for 30 min at 37°, without (CTRL) or with 1 μM arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), in the presence of E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme Ube2g2, ubiquitin and ATP. Samples were immunoprecipitated with an anti-HMGCoAR antibody; ubiquitinated HMGCoAR was measured by a chemiluminescence-based assay. Measurements were performed in triplicate and data are presented as means ± SD (n = 3). Versus CTRL HT29: * p < 0.005; versus CTRL HT29-dx: * p < 0.005. (B) HT29 and HT29-dx cells were incubated for 24 h in the absence (CTRL) or presence of 50 μM arachidonic acid (AA), docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA), then lysed and centrifuged to collect the microsomal fractions. Western blotting experiments were performed using anti-Insig-1, Insig-2, gp78, Trc8 antibodies. The expression of calreticulin (CRT) was measured to check the equal protein loading. The figure is representative of three experiments with similar results. The band density ratio between each protein and CRT was expressed as arbitrary units. Versus CTRL HT29: * p < 0.001. (C) Human recombinant HMGCoAR was incubated for 30 min at 37°, without (CTRL) or with various concentrations (1 nM, 10 nM, 100 nM, 1 μM) of arachidonic acid (AA), docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA), in the presence of E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme Ube2g2, E3 ligase enzymes gp78 or Trc8. The amount of ubiquitinated HMGCoAR was measured by a chemiluminescence-based assay. Measurements were performed in triplicate and data are presented as means ± SD (n = 3). Versus CTRL: * p < 0.05.