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. 2021 Aug 27;62:100112. doi: 10.1016/j.jlr.2021.100112

Fig. 6.

Fig. 6

Effect of ANGPTL8 R59W on EL and LPL inhibition. A: Western blot showing levels of ANGPTL3 and ANGPTL8 in conditioned media after transfection with ANGPTL3 alone (no A8) or cotransfected with wild-type ANGPTL8 (A8 WT) or ANGPTL8 R59W (A8 R59W). B: Phospholipase activity of EL after incubation for 30 min at 37°C with increasing concentrations of ANGPTL3, ANGPTL3-ANGPTL8 complexes, or ANGPTL3-ANGPTL8 (R59W) complexes. Points represent mean (±SD) of three independent experiments; each performed with biological duplicates. C: Phospholipase activity of EL-expressing RHMVECs after incubation with the indicated concentrations of ANGPTL3, ANGPTL3-ANGPTL8 complexes, or ANGPTL3-ANGPTL8 (R59W) complexes at 37°C for 30 min. Data points represent mean (±SD) of three independent experiments performed with biological triplicates. Activity was normalized to untreated EL-expressing RHMVECs. D: Relative protein binding of ANGPTL3, ANGPTL3-ANGPTL8 complexes, and ANGPTL3-ANGPTL8 (R59W) complexes as indicated by reconstituted luciferase activity. LargeBiT-EL was incubated with 0.6 μg/ml of smallBiT-tagged ANGPTL3, ANGPTL3-ANGPTL8 complexes ([A8] = 64 ng/ml), or ANGPTL3-ANGPTL8 (R59W) complexes ([A8] = 48.8 ng/ml) at 37°C for 30 min. Luminescence was measured after adding luciferase substrate. Bar graph represents mean (±SD) of three independent experiments performed with biological triplicates. E: Lipase activity of LPL after incubation for 30 min at 37°C with increasing concentrations of ANGPTL3, ANGPTL3-ANGPTL8 complexes, or ANGPTL3-ANGPTL8 (R59W) complexes. Points represent mean (±SD) of three independent experiments performed with biological duplicates. F: Relative protein binding of ANGPTL3, ANGPTL3-ANGPTL8 complexes, and ANGPTL3-ANGPTL8 (R59W) complexes as indicated by reconstituted luciferase activity. LargeBiT-LPL was bound to GPIHBP1-expressing RHMVECs. After washing off unbound LPL, 0.6 μg/ml of smallBiT-tagged ANGPTL3, ANGPTL3-ANGPTL8 complexes ([A8] = 64 ng/ml), or ANGPTL3-ANGPTL8 (R59W) complexes ([A8] = 48.8 ng/ml) were added, and luminescence was measured after adding luciferase substrate. Bar graph represents mean (±SD) of three independent experiments performed with biological triplicates. ANGPTL3, angiopoietin-like 3; ANGPTL8, angiopoietin-like 8; EL, endothelial lipase; RHMVECs, rat heart microvessel endothelial cells.