Fig 1: HDLHealthy, but not HDLCAD, decreased LTB4 production from macrophages through proteasome-mediated degradation of 5-LO. Macrophages (RAW 264.7 cell line, 1 × 106) were activated by ZyA (30 min at 37 °C), then treated with HDLHealthy or HDLCAD (10 μg protein). (a) LTB4 production from macrophages was quantified by LC/MS/MS. The results are expressed as pg/1 × 106 cells, mean ± SEM, N = 4-5 in each group. (b) Representative MRM-chromatograph and MS-MS spectrum are presented for the identification of LTB4. (c) Macrophage lysates were processed for western blot analysis of 5-LO. The results are shown as fold changes from N = 4 experiments. (d) 5-LO degradation by HDLHealthy is proteasome-dependent. ZyA-activated macrophages were pretreated for 30 min with the proteasome inhibitor MG 132 (1 µM) or vehicle, then incubated with HDLHealthy for 30 min at 37 °C. Lysates were collected for western blot analysis of 5-LO. The results are shown as the mean ± SEM from N = 4 experiments. *P < 0.05, **P < 0.01, ***P < 0.005. (e) HDLHealthy enhanced ubiquitination of 5-LO in macrophages. ZyA-activated macrophages were pretreated for 30 min with the proteasome inhibitor MG 132 (1 µM) or vehicle, then incubated with HDLHealthy or HDLCAD for 30 min at 37 °C. Lysates were immunoprecipitated with anti-5LO antibody (#3289 S, Cell Signaling), using a Dynabeads Protein A IP Kit (Thermo Fisher Scientific), and then immunoblotted with anti-ubiquitin antibody (ab140601, Abcam). (f) HDLHealthy enhanced 12/15-LO expression in macrophages. Macrophages (RAW 264.7 cell line, 1 × 106) were activated by ZyA (30 min at 37 °C), then treated with HDLHealthy or HDLCAD (10 μg protein, 30 min at 37 °C). After incubation, total RNA was extracted, and then, 12/15-LO expression was analysed by real-time PCR. The results are shown as fold change compared with the vehicle group; mean ± SEM, N = 4. *P < 0.05 vs the other groups.
Fig 2: Methylation of the Uba52 promoter and total K48 polyubiquitination levels are developmentally regulated within the amygdala of female rats. The second hemisphere of the basolateral amygdala (BLA) collected from the same animals used in Figs. 1 and 2 was used. Only a subset of the samples was used for the DNA methylation analyses. A Bisulfite sequencing was conducted to quantify methylation levels at CpG 4 of the Uba52 promoter. T-test analysis (indicated by $ symbol) within each sex was significant for female rats, with 9 wk females having increased methylation levels compared to 4 wk females (two-tailed t-test, t7 = 2.664, p = 0.0323), but was not significant in male rats (two-tailed t-test, t7 = 2.353, p = 0.0509). Group sizes are as follows: n = 5 in 4 wk male and female, n = 4 in 9 wk male and female. Two-way ANOVA (indicated by * symbol) was not significant for Age (F1,14 = 0.0936, p = 0.7642) or Sex (F1,14 = 1.061, p = 0.3204), but there was an interaction between Sex X Age (F1,14 = 12.61, p = 0.0032). Tukey’s honest significance difference post hoc test determined 9 wk females had higher methylation levels than 9 wk males (p = 0.0368). Group sizes are as follows: n = 5 in 4 wk male and female, n = 4 in 9 wk male and female. B Total K48-polyubiquitination levels were quantified using western blot. Two-tailed t-test (indicated by $ symbol) of males revealed no differences between 4 and 9 wk animals (two-tailed t-test, t18 = 0.1400, p = 0.8902), but 9 wk females had significantly higher K48-polyubiquitination levels compared to 4 wk females (two-tailed t-test, t17 = 3.190, p = 0.0054). Two-way ANOVA did not show significant differences for Age (F1,35 = 3.379, p = 0.0745), Sex (F1,35 = 2.457, p = 0.1260), or interaction between Sex X Age (F1,35 = 2.581, p = 0.1172). In representative western blot images, the top box is K48 polyubiquitination and the bottom box is β-actin, which was used as a loading control. Group sizes are as follows: n = 10 in 4 wk male, 9 wk male, and 9 wk female, n = 9 in 4 wk female. OD values were normalized to 4 wk males. *p < 0.05 in ANOVA and $p < 0.05 in t-test
Supplier Page from Abcam for Anti-Ubiquitin (linkage-specific K48) antibody [EP8589]