Fig 1: 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.
Fig 2: Secretion of ANGPTL8. (A) Western blots showing conditioned media (lanes 1–6) and cell lysates (lanes 7–12) from 293T cells transfected with full-length (F) or truncated (T) ANGPTL3 and/or with ANGPTL8. Blots on the right show media after 10-fold concentration using Millipore centrifugal filter units. (B) Western blots against ANGPTL8 showing cell lysates, conditioned media, and 10× concentrated conditioned media from HepG2 cells transfected with full-length ANGPTL3 and/or ANGPTL8. (C) Western blots showing cell lysate (lanes 1–3) and conditioned media (lanes 5–7) from 293T cells transfected with ANGPTL8 alone (lanes 1 and 5), ANGPTL8 alone and incubated in ANGPTL3 conditioned media for 48 h (lanes 2 and 6), or co-transfected with ANGPTL8 and ANGPTL3 (lanes 3 and 7). (D) Western blots showing conditioned media (lanes 1–6) and cell lysates (lanes 7–12) from 293T cells transfected with full-length ANGPTL3 (F), the C-terminal domain of ANGPTL3 (C) and/or with ANGPTL8. All western blots were performed with antibodies against the V5 tag for ANGPTL8 and against the Strep tag for ANGPTL3.
Fig 3: Pulldown of ANGPTL8 with ANGPTL3. (A–D) The conditioned media from cells transfected with Strep-ANGPTL3 (A), ANGPTL8 (B), or both Strep-ANGPTL3 and ANGPTL8 (C), as well as Strep-ANGPTL3 conditioned media mixed with ANGPTL8 conditioned media (D) were precipitated using Strep-tag antibody bound to protein A–coupled beads. (E–G) The lysates from cells transfected with Strep-ANGPTL3 (E), ANGPTL8 (F), or both Strep-ANGPTL3 and ANGPTL8 (G) were precipitated using Strep-tag antibody bound to protein A–coupled beads. (H–I) The conditioned media from cells transfected with V5-ANGPTL4 (H), or both V5-ANGPTL4 and Strep-ANGPTL3 (I) were precipitated using V5-tag antibody bound to protein G–coupled beads. All western blots show starting material (SM), unbound supernatant (Sup), washes, and elution (E).
Fig 4: ANGPTL3 inhibition of EL bound to cells or in the presence of heparin. A: Representative Western blot of media and lysate collected from rat heart microvessel endothelial cells (RHMVECs) expressing EL and treated with or without 0.1 U/ml heparin for 24 h. B: Phospholipase activity of media collected from EL-expressing RHMVECs treated with or without 0.1 U/ml heparin for 24 h. Points represent mean (±SD) of two independent experiments, each with three independent activity measurements. C: Phospholipase activity of EL-expressing RHMVECs after incubation with increasing concentrations of ANGPTL3 at 37°C for 30 min. Each data point represents mean (±SD) of three independent experiments performed with biological triplicates. Activity was normalized to untreated EL-expressing RHMVECs. D: Phospholipase activity of EL after incubation with (+ heparin (pre)) or without (- heparin) 1 U/ml heparin and with increasing concentrations of ANGPTL3 for 30 min at 37°C. Following incubation, a portion of the samples without heparin were then treated with 1 U/ml heparin (+ heparin (post)) and also assayed for phospholipase activity. Points represent mean (±SD) of three independent experiments performed with biological triplicates. E: Phospholipase activity over time of EL incubated with or without 1 U/ml heparin and with or without 2 µg/ml ANGPTL3 at 37°C. Points represent mean (±SD) of three independent experiments performed with biological triplicates. ANGPTL3, angiopoietin-like 3; EL, endothelial lipase.
Fig 5: Inhibition of endothelial lipase by ANGPTL3. A: Phospholipase activity of EL after incubation with increasing concentrations of ANGPTL3 for 30 min at 37°C. Activity was normalized to control-treated EL. Points represent mean (±SD) of three independent experiments; each performed with biological duplicates. B and C: Phospholipase activity over time of EL incubated with or without 2 µg/ml ANGPTL3. Activity was normalized either to the activity of EL without ANGPTL3 (control treated) at time point 0 (B) or to the respective control-treated EL for each time point (C). Points represent mean (±SD) of three independent experiments; each performed with biological duplicates. ANGPTL3, angiopoietin-like 3; EL, endothelial lipase.
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