anti human gpr109a  (R&D Systems)

Journal: Blood Advances

Article Title: HCAR2 is a novel receptor for heme ∗

doi: 10.1182/bloodadvances.2025016197

Figure Lengend Snippet: Functional validation of HCAR2 as a receptor for heme measured by receptor signaling. (A) Titration of heme in the activity assay in agonist mode of HCAR2/GPR109A. (B) Titration of nicotinic acid (niacin) in the activity assay in agonist mode of HCAR2/GPR109A.

Article Snippet: Cells were harvested and washed with PBS 1× and further stained with live-dead (Invitrogen; L10119A) and anti-human HCAR2 primary antibody (Novus; NBP1-92180) and labeled with chicken anti-rabbit AF647 secondary antibody (Invitrogen; A21443).

Techniques: Functional Assay, Biomarker Discovery, Titration, Activity Assay

Journal: Blood Advances

Article Title: HCAR2 is a novel receptor for heme ∗

doi: 10.1182/bloodadvances.2025016197

Figure Lengend Snippet: Biochemical validation of HCAR2 as a receptor for heme, illustrated by direct ligand/receptor binding. (A) Comparison by SPR of the interaction of immobilized recombinant HCAR2 compared to C3b (negative control) with different concentrations of heme (39 to 2500 nM). (B) Analysis of HCAR2-heme interaction by spectroscopy (0.35 to 10.2 μM). (C) Absorbance at the Soret peak (414 nM) as a function of the molar excess of heme over the protein. RU, response units.

Article Snippet: Cells were harvested and washed with PBS 1× and further stained with live-dead (Invitrogen; L10119A) and anti-human HCAR2 primary antibody (Novus; NBP1-92180) and labeled with chicken anti-rabbit AF647 secondary antibody (Invitrogen; A21443).

Techniques: Biomarker Discovery, Binding Assay, Comparison, Recombinant, Negative Control, Spectroscopy

Journal: Blood Advances

Article Title: HCAR2 is a novel receptor for heme ∗

doi: 10.1182/bloodadvances.2025016197

Figure Lengend Snippet: HCAR2 is overexpressed in hemolytic and SCD mice and its expression is regulated by heme/HO-1 axis. (A) Schematic overview of the experiment performed on mice. (B) Heat map of the normalized counts of the 3 main Hcar2 transcription factors of PHZ vs PBS (upper) and HbSS vs HbAA mice (lower). Normalized counts in each mouse are represented with row scale normalization. (C-F) Spearman correlation of Hcar2 and Hmox1 normalized gene expression levels in PHZ- and PBS-treated mice from RNAseq (C) and QuantiGene data (E) and HbSS and HbAA mice from RNAseq (D) and QuantiGene data (F). (G) QuantiGene analysis of Hcar2 gene expression levels in PHZ- and PBS-treated mice. (H) QuantiGene analysis of Hcar2 gene expression levels in HbSS and HbAA mice, either treated with PBS (left) or pretreated with SnMP, followed by an injection of 24-μM heme (right). (I-J) QuantiGene analysis of Hcar2 expression in HbAA mice injected with heme. (I) Level of expression of Hcar2 . (J) Spearman correlation of Hcar2 and Hmox1 normalized gene expression in the HbAA mice injected with PBS or heme. ∗∗∗∗ P < .0001; ∗ P < .05; Mann-Whitney test in panels G,I. ∗ P < .05; Kruskal-Wallis with Dunn test for multiple pairwise comparisons in panel H.

Article Snippet: Cells were harvested and washed with PBS 1× and further stained with live-dead (Invitrogen; L10119A) and anti-human HCAR2 primary antibody (Novus; NBP1-92180) and labeled with chicken anti-rabbit AF647 secondary antibody (Invitrogen; A21443).

Techniques: Expressing, Gene Expression, Injection, MANN-WHITNEY

Journal: British journal of pharmacology

Article Title: 4-Hydroxynonenal-induced GPR109A (HCA 2 receptor) activation elicits bipolar responses, G αi -mediated anti-inflammatory effects and G βγ -mediated cell death.

doi: 10.1111/bph.14174

Figure Lengend Snippet: Figure 1 GPR109A activation by 4-HNE, niacin and 3-OHBA induces apoptotic death in ARPE-19 and CCD-841 cells. (A) GPR109A and NOX4 expression levels were analysed by immunoblotting. Bar graphs represent averaged quantitations of immunoblots from six independent experiments. (B–F) Cell viability was measured via MTT assay. (G) ARPE-19 cells were stained with Hoechst 33258, a water-soluble DNA-binding compound, and 6-carboxyfluroscein diacetate, a secondary dye that accumulates in viable cells. (H) Annexin V-positive and PI-positive ARPE-19 cell popula- tions were determined via flow cytometry. (I) Caspase-3/7 activity was measured in 4-HNE-treated ARPE-19 cells using a Caspase-Glo® 3/7 assay kit. *P < 0.05 versus vehicle-treated controls.

Article Snippet: Monoclonal antibody against human GPR109A was obtained from Novus Biological (Cat. No. NBP2-12358AF488; Littleton, CO, USA).

Techniques: Activation Assay, Expressing, Western Blot, MTT Assay, Staining, Binding Assay, Cytometry, Activity Assay, Caspase-Glo Assay

Journal: British journal of pharmacology

Article Title: 4-Hydroxynonenal-induced GPR109A (HCA 2 receptor) activation elicits bipolar responses, G αi -mediated anti-inflammatory effects and G βγ -mediated cell death.

doi: 10.1111/bph.14174

Figure Lengend Snippet: Figure 7 4-HNE is a stronger agonist than niacin, not competing with niacin for binding sites in GPR109A. (A) A functional activity assay using the CHO-K1- GPR109A-Gi cell line, which was designed to detect the inhibition of cAMP generation, was performed in the cells pre-stimulated with forskolin prior to treatment with the other agonists. (B) 4-HNE, niacin, 3-OHBA did not induce cytotoxicity in CHO-K1-GPR109A-Gi cells. (C) SPR binding assay showing 4-HNE and niacin bind to GPR109A in a concentration-dependent manner. (D) [3H]-niacin binding competition assay with 4-HNE and niacin showing their effects are concentration-dependent in HEK-293T cells stably expressing human GPR109A. (E and F) CHO-K1 cells were transfected with five different FLAG-tagged plasmids of GPR109A mutants (R111A, R253A, W256A, F277A and L280A). (E) Indicates their trans- fection efficiency as demonstrated by FLAG staining and (F) shows the results of the functional activity assay indicating that 4-HNE and niacin de- creased the forskolin-induced production of cAMP in a concentration-dependent manner. (G–I) Effects of combination treatments with the GPR109A ligands (niacin, 3-OHBA or 4-HNE). (G) CHO-K1-GPR109A cells were treated with a combination of two from three ligands, and forskolin-induced cAMP levels were measured. In ARPE-19 cells, effects of combination treatments on changes in (H) intracellular Ca2+ levels and (I) cell viability were determined. *P < 0.05 versus vehicle-treated controls. #P < 0.05 versus niacin-treated cells. $P < 0.05 versus 3-OHBA- treated cells. &P < 0.05 versus 4-HNE-treated cells. RU, response units.

Article Snippet: Monoclonal antibody against human GPR109A was obtained from Novus Biological (Cat. No. NBP2-12358AF488; Littleton, CO, USA).

Techniques: Binding Assay, Functional Assay, Activity Assay, Inhibition, Concentration Assay, Competitive Binding Assay, Stable Transfection, Expressing, Transfection, Staining

Journal: Scientific Reports

Article Title: The effect of short-chain fatty acids on human monocyte-derived dendritic cells

doi: 10.1038/srep16148

Figure Lengend Snippet: ( a ) qPCR for SCFAs receptors on monocyte-derived DC compared to MCF-7 cell line as internal control. Shown are the averages ± standard deviations (SD) (n = 3). ( b ) Flow cytometry analysis of GPR41 and GPR109A surface expression on monocyte-derived DC and ( c ) primary CD141 + and CD1c + DC. Mean fluorescence intensity (MFI) averages ± standard deviations (SD) (n = 3) are reported below each histogram. Gates for GPR41- or GPR190A-positive cells in both figures ( b,c ) has been placed according to their respective isotype control. The different shades of grey represent cells from three different donors used for the experiments.

Article Snippet: Anti-human HLA-DR (PE, BD, clone L243), anti-human CD83 (BV421, Biolegend, clone HB15e) and anti-human CD86 (APC, Biolegend, clone IT2.2), anti-human CD141 (BV421, BD, clone 1A4), anti-human CD1c (BB515, BD, clone F10/21A3), anti-human GPR41 (APC, LSBio), anti-human GPR109A (APC, R&D), and all the respective isotypes were used (all purchased by BD or R&D).

Techniques: Derivative Assay, Control, Flow Cytometry, Expressing, Fluorescence