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R&D Systems n terminal region
N Terminal Region, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/af3829/Human+Angiopoietin-like+Protein+3%2FANGPTL3+Antibody/pmc12867014-306-9-13
Average 93 stars, based on 11 article reviews

n terminal region - by Bioz Stars, 2026-09

93/100 stars

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Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids
Article Snippet: Anti-human ANGPTL4 antibodies were generated by immunization with recombinant ANGPTL4 (residues 26-161), or purchased commercially (R&D Systems, AF3485). .. Antihuman ANGPTL3 antibodies were generated after immunization with mammalian produced recombinant ANGPTL3 (residues 17-220) or purchased commercially (R&D AF3829). .. Clones of interest were screened for non-overlapping epitopes, and antigen-specific variable heavy (VH) and light (VL) gene sequences were determined from extracted RNA using a mouse Ig primer set (EMD Millipore).

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids
Article Snippet: Anti-human ANGPTL4 antibodies were generated by immunization with recombinant ANGPTL4 (residues 26-161) or purchased commercially (R&D Systems, AF3485). .. Anti-human ANGPTL3 antibodies were generated after immunization with mammalian produced recombinant ANGPTL3 (residues 17-220) or purchased commercially (R&D Systems, AF3829). .. Clones of interest were screened for nonoverlapping epitopes, and antigen-specific variable heavy and variable light gene sequences were determined from extracted RNA using a mouse Ig primer set (EMD Millipore).

Produced:

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids
Article Snippet: Anti-human ANGPTL4 antibodies were generated by immunization with recombinant ANGPTL4 (residues 26-161) or purchased commercially (R&D Systems, AF3485). .. Anti-human ANGPTL3 antibodies were generated after immunization with mammalian produced recombinant ANGPTL3 (residues 17-220) or purchased commercially (R&D Systems, AF3829). .. Clones of interest were screened for nonoverlapping epitopes, and antigen-specific variable heavy and variable light gene sequences were determined from extracted RNA using a mouse Ig primer set (EMD Millipore).

Recombinant:

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids
Article Snippet: Anti-human ANGPTL4 antibodies were generated by immunization with recombinant ANGPTL4 (residues 26-161) or purchased commercially (R&D Systems, AF3485). .. Anti-human ANGPTL3 antibodies were generated after immunization with mammalian produced recombinant ANGPTL3 (residues 17-220) or purchased commercially (R&D Systems, AF3829). .. Clones of interest were screened for nonoverlapping epitopes, and antigen-specific variable heavy and variable light gene sequences were determined from extracted RNA using a mouse Ig primer set (EMD Millipore).

Image Search Results

Figure 1 Overexpression of ANGPTL3 inhibits IL-1β-induced NF-κB activation. (A

Journal: Journal of molecular cell biology

Article Title: ANGPTL3 negatively regulates IL-1β-induced NF-κB activation by inhibiting the IL1R1-associated signaling complex assembly.

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: Figure 1 Overexpression of ANGPTL3 inhibits IL-1β-induced NF-κB activation. (A

Article Snippet: Antibodies against ANGPTL3 (R&D Systems, AF3829), MyD88 (Cell Signaling Technology, 4283), Flag tag (Sigma, F1804), HA tag (Sigma, H3663), and Myc tag (Cell Signaling Technology, 2276) were purchased.

Techniques: Over Expression, Activation Assay

Figure 2 Knockdown of ANGPTL3 potentiates IL-1β-induced NF-κB activation. (A)

Journal: Journal of molecular cell biology

Article Title: ANGPTL3 negatively regulates IL-1β-induced NF-κB activation by inhibiting the IL1R1-associated signaling complex assembly.

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: Figure 2 Knockdown of ANGPTL3 potentiates IL-1β-induced NF-κB activation. (A)

Techniques: Knockdown, Activation Assay

Figure 3 Knockout of ANGPTL3 potentiates IL-1β-induced NF-κB activation. (A)

Journal: Journal of molecular cell biology

Article Title: ANGPTL3 negatively regulates IL-1β-induced NF-κB activation by inhibiting the IL1R1-associated signaling complex assembly.

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: Figure 3 Knockout of ANGPTL3 potentiates IL-1β-induced NF-κB activation. (A)

Techniques: Knock-Out, Activation Assay

Figure 4 ANGPTL3 targets the IL1R1-associated complex. (A) The effects of

Journal: Journal of molecular cell biology

Article Title: ANGPTL3 negatively regulates IL-1β-induced NF-κB activation by inhibiting the IL1R1-associated signaling complex assembly.

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: Figure 4 ANGPTL3 targets the IL1R1-associated complex. (A) The effects of

Techniques:

Figure 5 The intracellular FLD domain of ANGPTL3 is involved in the negative

Journal: Journal of molecular cell biology

Article Title: ANGPTL3 negatively regulates IL-1β-induced NF-κB activation by inhibiting the IL1R1-associated signaling complex assembly.

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: Figure 5 The intracellular FLD domain of ANGPTL3 is involved in the negative

Techniques:

Figure 6 ANGPTL3 inhibits the formation of IL1R1-associated complex. (A and B)

Journal: Journal of molecular cell biology

Article Title: ANGPTL3 negatively regulates IL-1β-induced NF-κB activation by inhibiting the IL1R1-associated signaling complex assembly.

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: Figure 6 ANGPTL3 inhibits the formation of IL1R1-associated complex. (A and B)

Techniques:

Figure 7 Intracellular ANGPTL3 inhibits the IL1R1–IL1RAP interaction to attenuate

Journal: Journal of molecular cell biology

Article Title: ANGPTL3 negatively regulates IL-1β-induced NF-κB activation by inhibiting the IL1R1-associated signaling complex assembly.

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: Figure 7 Intracellular ANGPTL3 inhibits the IL1R1–IL1RAP interaction to attenuate

Techniques:

Fig. 1. ANGPTL4/8-mediated plasmin generation blocks ANGPTL3/8-mediated LPL inhibition. A: LPL stable expres- sion cells were incubated with lipase substrate after being pre- viously incubated with vehicle alone, ANGPTL4/8, tPA + plasminogen, or ANGPTL4/8 + tPA + plasminogen. LPL ac- tivity was calculated as a percent of vehicle control. Results are shown as the mean ± SD (n = 10 from 4 independent experi- ments) (*P < 0.001 vs. vehicle). B: LPL stable expression cells were incubated with vehicle alone throughout the experiment or in the absence or presence of ANGPTL3/8 after being previously incubated with vehicle alone, ANGPTL4/8 alone, tPA + plasminogen, or ANGPTL4/8 + tPA + plasminogen. Afterward, LPL activity was assessed following the addition of fluorescent lipase substrate and calculated as a percent of vehicle control. Results are shown as the mean ± SD (n = 8 from three independent experiments) (*P < 0.001 vs. ANGPTL3/8 alone). C: Samples from Figure 1B that contained ANGPTL3/8 were assessed using ANGPTL3 antibody Western blotting. Re- sults are representative of three independent experiments, with two replicates for each experiment (total n = 6 from three in- dependent experiments). ANGPTL, angiopoietin-like protein; LPL, lipoprotein lipase; tPA, tissue plasminogen activator.

Journal: Journal of lipid research

Article Title: Decoding the role of angiopoietin-like protein 4/8 complex-mediated plasmin generation in the regulation of LPL activity.

doi: 10.1016/j.jlr.2023.100441

Figure Lengend Snippet: Fig. 1. ANGPTL4/8-mediated plasmin generation blocks ANGPTL3/8-mediated LPL inhibition. A: LPL stable expres- sion cells were incubated with lipase substrate after being pre- viously incubated with vehicle alone, ANGPTL4/8, tPA + plasminogen, or ANGPTL4/8 + tPA + plasminogen. LPL ac- tivity was calculated as a percent of vehicle control. Results are shown as the mean ± SD (n = 10 from 4 independent experi- ments) (*P < 0.001 vs. vehicle). B: LPL stable expression cells were incubated with vehicle alone throughout the experiment or in the absence or presence of ANGPTL3/8 after being previously incubated with vehicle alone, ANGPTL4/8 alone, tPA + plasminogen, or ANGPTL4/8 + tPA + plasminogen. Afterward, LPL activity was assessed following the addition of fluorescent lipase substrate and calculated as a percent of vehicle control. Results are shown as the mean ± SD (n = 8 from three independent experiments) (*P < 0.001 vs. ANGPTL3/8 alone). C: Samples from Figure 1B that contained ANGPTL3/8 were assessed using ANGPTL3 antibody Western blotting. Re- sults are representative of three independent experiments, with two replicates for each experiment (total n = 6 from three in- dependent experiments). ANGPTL, angiopoietin-like protein; LPL, lipoprotein lipase; tPA, tissue plasminogen activator.

Article Snippet: Human plasminogen (1939-SE), anti-ApoC2 rabbit antibody (MAF4497), anti-ANGPTL3 rabbit polyclonal antibody (AF3829), and anti-LPL antibody (AF7197) were purchased from R&D systems.

Techniques: Inhibition, Incubation, Control, Expressing, Activity Assay, Western Blot

Fig. 2. ANGPTL4/8-mediated plasmin generation blocks LPL inhibition by ANGPTL4 and ANGPTL3. A: LPL stable expression cells were incubated with vehicle alone throughout the experiment or in the absence or presence of ANGPTL4 after being previously preincubated with vehicle alone, ANGPTL4/8 alone, tPA + plasminogen, or ANGPTL4/8 + tPA + plasminogen. Afterward, LPL activity was assessed following the addition of fluorescent lipase substrate and calculated as a percent of vehicle control. Results are shown as the mean ± SD (n = 6 from two independent experiments) (*P < 0.001 vs. ANGPTL4 alone). B: LPL stable expression cells were incubated with vehicle alone throughout the experiment or in the absence or presence of ANGPTL3 after being previously preincubated with vehicle alone, ANGPTL4/8 alone, tPA + plasminogen, or ANGPTL4/8 + tPA + plasminogen. After- ward, LPL activity was assessed following the addition of fluo- rescent lipase substrate and calculated as a percent of vehicle control. Results are shown as the mean ± SD (n = 4 from two independent experiments) (*P < 0.001 vs. ANGPTL3 alone). C: Samples from Figure 2B that contained ANGPTL3 were

Journal: Journal of lipid research

Article Title: Decoding the role of angiopoietin-like protein 4/8 complex-mediated plasmin generation in the regulation of LPL activity.

doi: 10.1016/j.jlr.2023.100441

Figure Lengend Snippet: Fig. 2. ANGPTL4/8-mediated plasmin generation blocks LPL inhibition by ANGPTL4 and ANGPTL3. A: LPL stable expression cells were incubated with vehicle alone throughout the experiment or in the absence or presence of ANGPTL4 after being previously preincubated with vehicle alone, ANGPTL4/8 alone, tPA + plasminogen, or ANGPTL4/8 + tPA + plasminogen. Afterward, LPL activity was assessed following the addition of fluorescent lipase substrate and calculated as a percent of vehicle control. Results are shown as the mean ± SD (n = 6 from two independent experiments) (*P < 0.001 vs. ANGPTL4 alone). B: LPL stable expression cells were incubated with vehicle alone throughout the experiment or in the absence or presence of ANGPTL3 after being previously preincubated with vehicle alone, ANGPTL4/8 alone, tPA + plasminogen, or ANGPTL4/8 + tPA + plasminogen. After- ward, LPL activity was assessed following the addition of fluo- rescent lipase substrate and calculated as a percent of vehicle control. Results are shown as the mean ± SD (n = 4 from two independent experiments) (*P < 0.001 vs. ANGPTL3 alone). C: Samples from Figure 2B that contained ANGPTL3 were

Techniques: Inhibition, Expressing, Incubation, Activity Assay, Control

Fig. 5. Characterization of ANGPTL4/8-mediated plasmin generation and its effects on LPL. A: The ability of tPA to convert plasminogen to plasmin in the presence of ANGPTL4/8 was examined after addition of ANGPTL3/8, ANGPTL3, ANGPTL4, ApoC3, or ApoC2, with the molar ratio of each protein to ANGPTL4/8 being the same as in the LPL activity assays. Generation of plasmin was measured us- ing colorimetric plasmin activity assays, with absorbances recorded spectrophotometrically. Results shown are repre- sentative of three independent experiments. B: LPL stable–expressing cells were preincubated with ANGPTL4/8, followed by incubation in the absence or presence of tPA + plasminogen. Cells were washed, and heparin was added to release membrane-bound LPL, which was measured via Western blotting. Results are presentative of 6 replicates from 2 independent experiments. C: Following removal of membrane-bound LPL, cells were solubilized using RIPA buffer, and LPL present within the cells was analyzed via Western blotting. Results are presentative of six replicates from two independent experiments. ANGPTL, angiopoietin- like protein; LPL, lipoprotein lipase; tPA, tissue plasminogen activator.

Journal: Journal of lipid research

Article Title: Decoding the role of angiopoietin-like protein 4/8 complex-mediated plasmin generation in the regulation of LPL activity.

doi: 10.1016/j.jlr.2023.100441

Figure Lengend Snippet: Fig. 5. Characterization of ANGPTL4/8-mediated plasmin generation and its effects on LPL. A: The ability of tPA to convert plasminogen to plasmin in the presence of ANGPTL4/8 was examined after addition of ANGPTL3/8, ANGPTL3, ANGPTL4, ApoC3, or ApoC2, with the molar ratio of each protein to ANGPTL4/8 being the same as in the LPL activity assays. Generation of plasmin was measured us- ing colorimetric plasmin activity assays, with absorbances recorded spectrophotometrically. Results shown are repre- sentative of three independent experiments. B: LPL stable–expressing cells were preincubated with ANGPTL4/8, followed by incubation in the absence or presence of tPA + plasminogen. Cells were washed, and heparin was added to release membrane-bound LPL, which was measured via Western blotting. Results are presentative of 6 replicates from 2 independent experiments. C: Following removal of membrane-bound LPL, cells were solubilized using RIPA buffer, and LPL present within the cells was analyzed via Western blotting. Results are presentative of six replicates from two independent experiments. ANGPTL, angiopoietin- like protein; LPL, lipoprotein lipase; tPA, tissue plasminogen activator.

Techniques: Activity Assay, Expressing, Incubation, Membrane, Western Blot

Fig. 6. An updated model for adipose tissue LPL regulation in the postprandial state. After feeding, increased ANGPTL4/8 (a) binds LPL, protects it from inactivation by ANGPTL4, but also partially inhibits LPL (light blue color) (b). Partially active ANGPTL4/8-LPL binds GPIHBP1 on abluminal endothelial surfaces (c) and is translocated (d). Concurrently, tPA secreted by the endothelium and plasminogen present on endothelial plasminogen receptors (PLG-R) bind the LPL-ANGPTL4/8 complex on luminal capillary surfaces (e). ANGPTL4/8 increases tPA-mediated generation of plasmin, which cleaves ANGPTL4/8 (f) to restore LPL activity (dark blue color). The plasmin generated (g) also protects LPL from inhibition by circulating ANGPTL3/8, ANGPTL3, and ApoC3 as well as from any localized ANGPTL4 that may be present (for the purpose of clarity only ANGPTL3/8 is shown in the figure) (h), while permitting ApoC2-mediated LPL stimulation to occur (i). ANGPTL, angiopoietin-like protein; LPL, lipoprotein lipase; tPA, tissue plasminogen activator.

Journal: Journal of lipid research

Article Title: Decoding the role of angiopoietin-like protein 4/8 complex-mediated plasmin generation in the regulation of LPL activity.

doi: 10.1016/j.jlr.2023.100441

Figure Lengend Snippet: Fig. 6. An updated model for adipose tissue LPL regulation in the postprandial state. After feeding, increased ANGPTL4/8 (a) binds LPL, protects it from inactivation by ANGPTL4, but also partially inhibits LPL (light blue color) (b). Partially active ANGPTL4/8-LPL binds GPIHBP1 on abluminal endothelial surfaces (c) and is translocated (d). Concurrently, tPA secreted by the endothelium and plasminogen present on endothelial plasminogen receptors (PLG-R) bind the LPL-ANGPTL4/8 complex on luminal capillary surfaces (e). ANGPTL4/8 increases tPA-mediated generation of plasmin, which cleaves ANGPTL4/8 (f) to restore LPL activity (dark blue color). The plasmin generated (g) also protects LPL from inhibition by circulating ANGPTL3/8, ANGPTL3, and ApoC3 as well as from any localized ANGPTL4 that may be present (for the purpose of clarity only ANGPTL3/8 is shown in the figure) (h), while permitting ApoC2-mediated LPL stimulation to occur (i). ANGPTL, angiopoietin-like protein; LPL, lipoprotein lipase; tPA, tissue plasminogen activator.

Techniques: Activity Assay, Generated, Inhibition

ANGPTL3/8 and ANGPTL4/8 complexes increase with feeding. A: Recombinant human ANGPTL proteins and complexes used for immunoassays were characterized via electrophoresis. One microgram of each recombinant protein or complex was analyzed using gradient gel electrophoresis with a 4–20% Tris-glycine gel, followed by Coomassie Blue staining. B: Active ANGPTL4 (defined as full-length ANGPTL4 or the N-terminal fragment of ANGPTL4), CTDC ANGPTL4, ANGPTL3, ANGPTL8, ANGPTL3/8, and ANGPTL4/8 were measured in 50 normal donors using dedicated sandwich immunoassays. C: ANGPTL3/8, ANGPTL4/8, ANGPTL3, and ANGPTL8 were measured using dedicated sandwich immunoassays in 10 normal donors during fasting conditions and 1 and 2 h following a mixed meal challenge. Results are shown as the mean ± SEM. Significance for the feeding effect on ANGPTL proteins and complexes was assessed using a paired t -test.

Journal: Journal of Lipid Research

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids [S]

doi: 10.1194/jlr.RA120000781

Figure Lengend Snippet: ANGPTL3/8 and ANGPTL4/8 complexes increase with feeding. A: Recombinant human ANGPTL proteins and complexes used for immunoassays were characterized via electrophoresis. One microgram of each recombinant protein or complex was analyzed using gradient gel electrophoresis with a 4–20% Tris-glycine gel, followed by Coomassie Blue staining. B: Active ANGPTL4 (defined as full-length ANGPTL4 or the N-terminal fragment of ANGPTL4), CTDC ANGPTL4, ANGPTL3, ANGPTL8, ANGPTL3/8, and ANGPTL4/8 were measured in 50 normal donors using dedicated sandwich immunoassays. C: ANGPTL3/8, ANGPTL4/8, ANGPTL3, and ANGPTL8 were measured using dedicated sandwich immunoassays in 10 normal donors during fasting conditions and 1 and 2 h following a mixed meal challenge. Results are shown as the mean ± SEM. Significance for the feeding effect on ANGPTL proteins and complexes was assessed using a paired t -test.

Article Snippet: For ANGPTL3, a monoclonal antibody was used for capture and a polyclonal anti-ANGPTL3 antibody (R&D Systems, AF3829) was utilized for detection.

Techniques: Recombinant, Electrophoresis, Nucleic Acid Electrophoresis, Staining

ANGPTL8 circulates in ANGPTL3/8 and ANGPTL4/8 complexes. A: Anti-ANGPTL8, anti-ANGPTL4, and anti-ANGPTL3 antibodies covalently coupled to beads, with heavy and light chains further cross-linked, were used to immunoprecipitate (IP) human serum. Proteins were separated on a 12% Bis-Tris gel and transferred to PVDF. Co-immunoprecipitating proteins were visualized via Western blotting. Results are representative of two independent experiments. B: ANGPTL8, ANGPTL4, and ANGPTL3 were immunoprecipitated from human serum. Beads were washed using PBS, and bound proteins were reduced with DTT and alkylated. Following digestion, digests were acidified, and co-immunoprecipitating proteins were quantified using a mass spectrometry LC-MRM method. Results are shown as the mean ± SEM (n = 3) from one experiment representative of two independent experiments.

Journal: Journal of Lipid Research

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids [S]

doi: 10.1194/jlr.RA120000781

Figure Lengend Snippet: ANGPTL8 circulates in ANGPTL3/8 and ANGPTL4/8 complexes. A: Anti-ANGPTL8, anti-ANGPTL4, and anti-ANGPTL3 antibodies covalently coupled to beads, with heavy and light chains further cross-linked, were used to immunoprecipitate (IP) human serum. Proteins were separated on a 12% Bis-Tris gel and transferred to PVDF. Co-immunoprecipitating proteins were visualized via Western blotting. Results are representative of two independent experiments. B: ANGPTL8, ANGPTL4, and ANGPTL3 were immunoprecipitated from human serum. Beads were washed using PBS, and bound proteins were reduced with DTT and alkylated. Following digestion, digests were acidified, and co-immunoprecipitating proteins were quantified using a mass spectrometry LC-MRM method. Results are shown as the mean ± SEM (n = 3) from one experiment representative of two independent experiments.

Article Snippet: For ANGPTL3, a monoclonal antibody was used for capture and a polyclonal anti-ANGPTL3 antibody (R&D Systems, AF3829) was utilized for detection.

Techniques: Western Blot, Immunoprecipitation, Mass Spectrometry

Determination of ANGPTL3/8 and ANGPTL4/8 protein ratios by mass spectrometry

Journal: Journal of Lipid Research

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids [S]

doi: 10.1194/jlr.RA120000781

Figure Lengend Snippet: Determination of ANGPTL3/8 and ANGPTL4/8 protein ratios by mass spectrometry

Article Snippet: For ANGPTL3, a monoclonal antibody was used for capture and a polyclonal anti-ANGPTL3 antibody (R&D Systems, AF3829) was utilized for detection.

Techniques:

Circulating ANGPTL3/8 and ANGPTL4/8 are highly correlated with metabolic syndrome markers

Journal: Journal of Lipid Research

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids [S]

doi: 10.1194/jlr.RA120000781

Figure Lengend Snippet: Circulating ANGPTL3/8 and ANGPTL4/8 are highly correlated with metabolic syndrome markers

Article Snippet: For ANGPTL3, a monoclonal antibody was used for capture and a polyclonal anti-ANGPTL3 antibody (R&D Systems, AF3829) was utilized for detection.

Techniques:

ANGPTL3/8 blocks LPL-facilitated hepatocyte VLDL-C uptake. Cholesterol uptake in Huh7 hepatocytes was measured in the absence or presence of LPL pre-incubated with vehicle, ANGPTL3/8 complex, or ANGPTL4/8 complex for 1 h before mixing with fluorescent-labeled VLDL, followed by addition to the Huh7 hepatocytes for 30 min. The media was then replaced with fixative. Cells were fixed for 20 min, washed twice with PBS, and covered with PBS. Fluorescence at 495/525 nm was measured, with VLDL uptake calculated as relative fluorescent units at 525 nm. Results are shown as the mean ± SEM (n = 3).

Journal: Journal of Lipid Research

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids [S]

doi: 10.1194/jlr.RA120000781

Figure Lengend Snippet: ANGPTL3/8 blocks LPL-facilitated hepatocyte VLDL-C uptake. Cholesterol uptake in Huh7 hepatocytes was measured in the absence or presence of LPL pre-incubated with vehicle, ANGPTL3/8 complex, or ANGPTL4/8 complex for 1 h before mixing with fluorescent-labeled VLDL, followed by addition to the Huh7 hepatocytes for 30 min. The media was then replaced with fixative. Cells were fixed for 20 min, washed twice with PBS, and covered with PBS. Fluorescence at 495/525 nm was measured, with VLDL uptake calculated as relative fluorescent units at 525 nm. Results are shown as the mean ± SEM (n = 3).

Article Snippet: For ANGPTL3, a monoclonal antibody was used for capture and a polyclonal anti-ANGPTL3 antibody (R&D Systems, AF3829) was utilized for detection.

Techniques: Incubation, Labeling, Fluorescence

ANGPTL3/8 and ANGPTL4/8 manifest different binding patterns to LPL. A: The ability of ANGPTL3 and ANGPTL3/8 to bind LPL was assessed with bio-layer interferometry. Avidin-tagged LPL was immobilized on streptavidin biosensors and incubated with ANGPTL3 or ANGPTL3/8 and transferred to buffer-only wells to monitor dissociation. The left side of the graph shows the association of ANGPTL3 and ANGPTL3/8 with LPL. The right side shows their respective dissociations. Results are representative of three independent experiments. B: The ability of ANGPTL4 and ANGPTL4/8 to bind LPL was assessed with bio-layer interferometry. Avidin-tagged LPL was immobilized on streptavidin biosensors and incubated with ANGPTL4 or ANGPTL4/8 and transferred to buffer-only wells to monitor dissociation. The left side of the graph shows the association of ANGPTL4 and ANGPTL4/8 with LPL. The right side shows their respective dissociations. Results are representative of three independent experiments.

Journal: Journal of Lipid Research

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids [S]

doi: 10.1194/jlr.RA120000781

Figure Lengend Snippet: ANGPTL3/8 and ANGPTL4/8 manifest different binding patterns to LPL. A: The ability of ANGPTL3 and ANGPTL3/8 to bind LPL was assessed with bio-layer interferometry. Avidin-tagged LPL was immobilized on streptavidin biosensors and incubated with ANGPTL3 or ANGPTL3/8 and transferred to buffer-only wells to monitor dissociation. The left side of the graph shows the association of ANGPTL3 and ANGPTL3/8 with LPL. The right side shows their respective dissociations. Results are representative of three independent experiments. B: The ability of ANGPTL4 and ANGPTL4/8 to bind LPL was assessed with bio-layer interferometry. Avidin-tagged LPL was immobilized on streptavidin biosensors and incubated with ANGPTL4 or ANGPTL4/8 and transferred to buffer-only wells to monitor dissociation. The left side of the graph shows the association of ANGPTL4 and ANGPTL4/8 with LPL. The right side shows their respective dissociations. Results are representative of three independent experiments.

Article Snippet: For ANGPTL3, a monoclonal antibody was used for capture and a polyclonal anti-ANGPTL3 antibody (R&D Systems, AF3829) was utilized for detection.

Techniques: Binding Assay, Avidin-Biotin Assay, Incubation

LPL-binding characteristics of ANGPTL proteins and complexes

Journal: Journal of Lipid Research

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids [S]

doi: 10.1194/jlr.RA120000781

Figure Lengend Snippet: LPL-binding characteristics of ANGPTL proteins and complexes

Article Snippet: For ANGPTL3, a monoclonal antibody was used for capture and a polyclonal anti-ANGPTL3 antibody (R&D Systems, AF3829) was utilized for detection.

Techniques:

ANGPTL8 markedly increases ANGPTL3 inhibition of LPL but dramatically decreases ANGPTL4 inhibition of LPL. A: The ability of ANGPTL3 or ANGPTL3/8 to inhibit LPL was assessed using LPL-stable expression cells incubated with ANGPTL3 or ANGPTL3/8 prior to the addition of lipase substrate. Fluorescence was monitored at 1 and 30 min to correct for background. ANGPTL3/8 showed a 186-fold increase in LPL inhibition compared to ANGPTL3 alone (IC 50 values of 0.14 nM versus 26 nM, respectively). Results are shown as the mean ± SEM (n = 5). B: The ability of ANGPTL4 or ANGPTL4/8 to inhibit LPL was similarly assessed. ANGPTL4/8 showed a 128-fold decrease in LPL inhibition compared to ANGPTL4 alone (IC 50 values of 37 nM versus 0.29 nM, respectively). Results are shown as the mean ± SEM (n = 6). C: ANGPTL4 or ANGPTL4/8 inhibition of LPL was assessed using VLDL as a substrate. The assay was similar to that used in A and B, except that lipase substrate was replaced with VLDL and free FAs were measured. ANGPTL4/8 showed a 239-fold decrease in LPL inhibition compared to ANGPTL4 alone (IC 50 values of 105 nM versus 0.44 nM, respectively). Results are shown as the mean ± SEM (n = 3).

Journal: Journal of Lipid Research

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids [S]

doi: 10.1194/jlr.RA120000781

Figure Lengend Snippet: ANGPTL8 markedly increases ANGPTL3 inhibition of LPL but dramatically decreases ANGPTL4 inhibition of LPL. A: The ability of ANGPTL3 or ANGPTL3/8 to inhibit LPL was assessed using LPL-stable expression cells incubated with ANGPTL3 or ANGPTL3/8 prior to the addition of lipase substrate. Fluorescence was monitored at 1 and 30 min to correct for background. ANGPTL3/8 showed a 186-fold increase in LPL inhibition compared to ANGPTL3 alone (IC 50 values of 0.14 nM versus 26 nM, respectively). Results are shown as the mean ± SEM (n = 5). B: The ability of ANGPTL4 or ANGPTL4/8 to inhibit LPL was similarly assessed. ANGPTL4/8 showed a 128-fold decrease in LPL inhibition compared to ANGPTL4 alone (IC 50 values of 37 nM versus 0.29 nM, respectively). Results are shown as the mean ± SEM (n = 6). C: ANGPTL4 or ANGPTL4/8 inhibition of LPL was assessed using VLDL as a substrate. The assay was similar to that used in A and B, except that lipase substrate was replaced with VLDL and free FAs were measured. ANGPTL4/8 showed a 239-fold decrease in LPL inhibition compared to ANGPTL4 alone (IC 50 values of 105 nM versus 0.44 nM, respectively). Results are shown as the mean ± SEM (n = 3).

Article Snippet: For ANGPTL3, a monoclonal antibody was used for capture and a polyclonal anti-ANGPTL3 antibody (R&D Systems, AF3829) was utilized for detection.

Techniques: Inhibition, Expressing, Incubation, Fluorescence

LPL inhibition summary for ANGPTL3 versus ANGPTL3/8 and ANGPTL4 versus ANGPTL4/8 activity assays

Journal: Journal of Lipid Research

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids [S]

doi: 10.1194/jlr.RA120000781

Figure Lengend Snippet: LPL inhibition summary for ANGPTL3 versus ANGPTL3/8 and ANGPTL4 versus ANGPTL4/8 activity assays

Article Snippet: For ANGPTL3, a monoclonal antibody was used for capture and a polyclonal anti-ANGPTL3 antibody (R&D Systems, AF3829) was utilized for detection.

Techniques: Inhibition, Activity Assay

ANGPTL4/8 blocks ANGPTL3/8- and ANGPTL4-mediated inhibition of LPL. A: To study the ability of ANGPTL4/8 to protect LPL from ANGPTL3/8 inhibition, various concentrations of ANGPTL4/8 were pre-incubated with LPL-stable expression cells for 1 h. Afterward, 1 nM of ANGPTL3/8 was added for a further 1 h incubation, prior to the addition of lipase substrate. Fluorescence was monitored as in . Results are shown as the mean ± SEM (n = 4). B: To study the ability of ANGPTL4/8 to protect LPL from ANGPTL4 inhibition, various concentrations of ANGPTL4/8 were pre-incubated with LPL-stable expression cells for 1 h. Afterward, 1 nM of ANGPTL4 was added for a further 1 h incubation, prior to the addition of lipase substrate. Fluorescence was monitored as in . Results are shown as the mean ± SEM (n = 6).

Journal: Journal of Lipid Research

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids [S]

doi: 10.1194/jlr.RA120000781

Figure Lengend Snippet: ANGPTL4/8 blocks ANGPTL3/8- and ANGPTL4-mediated inhibition of LPL. A: To study the ability of ANGPTL4/8 to protect LPL from ANGPTL3/8 inhibition, various concentrations of ANGPTL4/8 were pre-incubated with LPL-stable expression cells for 1 h. Afterward, 1 nM of ANGPTL3/8 was added for a further 1 h incubation, prior to the addition of lipase substrate. Fluorescence was monitored as in . Results are shown as the mean ± SEM (n = 4). B: To study the ability of ANGPTL4/8 to protect LPL from ANGPTL4 inhibition, various concentrations of ANGPTL4/8 were pre-incubated with LPL-stable expression cells for 1 h. Afterward, 1 nM of ANGPTL4 was added for a further 1 h incubation, prior to the addition of lipase substrate. Fluorescence was monitored as in . Results are shown as the mean ± SEM (n = 6).

Article Snippet: For ANGPTL3, a monoclonal antibody was used for capture and a polyclonal anti-ANGPTL3 antibody (R&D Systems, AF3829) was utilized for detection.

Techniques: Inhibition, Incubation, Expressing, Fluorescence

Insulin stimulates human hepatocyte secretion of ANGPTL3/8. A: Insulin-naïve patients (n = 279) were administered the hepatic-preferential insulin BIL, and serum samples were obtained under morning fasting conditions over the course of 1 year of BIL treatment. ANGPTL3/8 and ANGPTL4/8 levels were measured at baseline and after 12, 26, and 52 weeks of BIL administration. Results are shown as the mean ± SEM (* P < 0.0001 versus week 0). B: Human primary hepatocytes obtained in the HepatoPac platform were washed in serum-free application media and pre-incubated in application media in the absence of insulin. Following aspiration, cells were incubated with application media in the absence or presence of 1 nM of insulin. ANGPTL3/8 and ANGPTL4/8 levels in the media were measured using sandwich immunoassays, with the results shown as the mean ± SEM (n = 8).

Journal: Journal of Lipid Research

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids [S]

doi: 10.1194/jlr.RA120000781

Figure Lengend Snippet: Insulin stimulates human hepatocyte secretion of ANGPTL3/8. A: Insulin-naïve patients (n = 279) were administered the hepatic-preferential insulin BIL, and serum samples were obtained under morning fasting conditions over the course of 1 year of BIL treatment. ANGPTL3/8 and ANGPTL4/8 levels were measured at baseline and after 12, 26, and 52 weeks of BIL administration. Results are shown as the mean ± SEM (* P < 0.0001 versus week 0). B: Human primary hepatocytes obtained in the HepatoPac platform were washed in serum-free application media and pre-incubated in application media in the absence of insulin. Following aspiration, cells were incubated with application media in the absence or presence of 1 nM of insulin. ANGPTL3/8 and ANGPTL4/8 levels in the media were measured using sandwich immunoassays, with the results shown as the mean ± SEM (n = 8).

Article Snippet: For ANGPTL3, a monoclonal antibody was used for capture and a polyclonal anti-ANGPTL3 antibody (R&D Systems, AF3829) was utilized for detection.

Techniques: Incubation

A possible model for how ANGPTL8 shifts FA toward adipose tissue after feeding. A: While fasting, ANGPTL8 levels are low. Localized ANGPTL4 inhibits adipose tissue LPL to minimize FA uptake into the fat for storage, and FAs are mainly taken up into skeletal muscle for use as energy. B: During feeding, ANGPTL8 forms a circulating complex with ANGPTL3 that increases its ability to inhibit LPL, thus minimizing FA uptake into skeletal muscle. ANGPTL8 also forms a mostly localized complex with ANGPTL4 in adipose tissue that decreases the ability of ANGPTL4 to inhibit LPL. The ANGPTL4/8 complex also protects LPL in the fat from inhibition by circulating ANGPTL3/8 and localized ANGPTL4 (denoted by red Xs), thereby preserving adipose tissue LPL activity to promote FA uptake into the fat for storage as TG.

Journal: Journal of Lipid Research

Article Title: Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids [S]

doi: 10.1194/jlr.RA120000781

Figure Lengend Snippet: A possible model for how ANGPTL8 shifts FA toward adipose tissue after feeding. A: While fasting, ANGPTL8 levels are low. Localized ANGPTL4 inhibits adipose tissue LPL to minimize FA uptake into the fat for storage, and FAs are mainly taken up into skeletal muscle for use as energy. B: During feeding, ANGPTL8 forms a circulating complex with ANGPTL3 that increases its ability to inhibit LPL, thus minimizing FA uptake into skeletal muscle. ANGPTL8 also forms a mostly localized complex with ANGPTL4 in adipose tissue that decreases the ability of ANGPTL4 to inhibit LPL. The ANGPTL4/8 complex also protects LPL in the fat from inhibition by circulating ANGPTL3/8 and localized ANGPTL4 (denoted by red Xs), thereby preserving adipose tissue LPL activity to promote FA uptake into the fat for storage as TG.

Article Snippet: For ANGPTL3, a monoclonal antibody was used for capture and a polyclonal anti-ANGPTL3 antibody (R&D Systems, AF3829) was utilized for detection.

Techniques: Inhibition, Preserving, Activity Assay

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