anti hcar2  (Novus Biologicals)

Journal: Cell reports

Article Title: Molecular recognition of niacin and lipid-lowering drugs by the human hydroxycarboxylic acid receptor 2.

doi: 10.1016/j.celrep.2023.113406

Figure Lengend Snippet: Figure 7. Mechanism of HCAR2-Gi coupling (A) Structural comparison of the GSK256073- bound HCAR2-Gi complex with representative class A Gi-coupled D2R (PDB: 7JVR). (B and C) Detailed interactions between Gai and HCAR2. The polar interactions are depicted by black dashed lines. See also Figure S9.

Article Snippet: The HCAR2-Gi complexes were formed in membranes by the addition of 40mM Niacin (TargetMol), or 20mM MK-6892 (TargetMol), or 40mM GSK256073 (TargetMol), respectively.

Techniques: Comparison

Journal: Clinical and Translational Medicine

Article Title: Niacin promotes motor function recovery after spinal cord injury via Hcar2‐dependent microglia immunometabolic regulation

doi: 10.1002/ctm2.70683

Figure Lengend Snippet: Single‐cell RNA sequencing showed cellular heterogeneity in the injured spinal cord and identified Hcar2 as a highly expressed gene in activated microglia following SCI. (A) Schematic of the bioinformatic workflow, which integrates public scRNA‐seq data (GEO: GSE162610 ) and employs nonnegative matrix factorization (NMF) and pseudobulk differential gene expression (DEG) analysis. (B) UMAP visualization of 12 distinct cell types identified from 59 040 cells in the mouse spinal cord. The cell clusters are color‐coded by annotation. (C) NMF consensus matrix heatmap identifying 10 stable gene meta‐programs (MPs). MP3, the immune activation module, is highlighted. (D) Gene Ontology (GO) enrichment heatmap for genes within the top MP. MP3 is highly enriched in neuroinflammatory and immune response pathways. (E) Volcano plot of DEGs (SCI vs sham). The upregulated genes ( n = 373; adjusted p < .001, log2FC > 1) are red; the downregulated genes ( n = 297; adjusted p < .001, log2FC < ‐1) are blue. (F) Venn diagram illustrating the intersection of significantly upregulated DEGs and the immune meta‐programme (MP3) signature. (G) Key genes, including Hcar2 , identified at the intersection of MP3 and upregulated DEGs, contributing to the ‘immune score’.

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated overnight at 4°C with the indicated primary antibodies: against Hcar2 (1:2000; AWA45885 ; Abiowell), Arg‐1 (1:2000; sc‐271430; Santa Cruz Biotechnology), CD206 (1:2000; ET1702‐04; HUABIO), TGF‐β (1:2000; AWA10316 ; Abiowell), or HRP‐conjugated β‐actin (1:10000; 700068; Zenbio).

Techniques: Single Cell, RNA Sequencing, Gene Expression, Activation Assay

Journal: Clinical and Translational Medicine

Article Title: Niacin promotes motor function recovery after spinal cord injury via Hcar2‐dependent microglia immunometabolic regulation

doi: 10.1002/ctm2.70683

Figure Lengend Snippet: Hcar2 was specifically upregulated in an activated microglial subtype (AM3) following SCI. (A) UMAP plot showing Hcar2 expression, primarily localized to immune cell clusters. (B) Kernel density plot confirming high Hcar2 expression density within microglia and neutrophil populations. (C) Dotplot illustrating Hcar2 expression levels and percentage of expression across all identified cell types at 1‐, 3‐, and 7‐day post‐injury (dpi) compared to sham controls. (D) UMAP plot of the reclustered microglial population, identifying 7 distinct subtypes: homeostatic microglia (HM), differentiating microglia (DM), activated microglia (AM1‐3), and interferon‐related microglia (IRM). (E and F) Ridge plot (E) and density plot overlay (F) showing Hcar2 expression is highly and specifically concentrated within the AM3 microglial subtype. (G) Heatmap displaying scaled expression of selected marker genes defining each microglial subtype. (H and I) Pseudotime trajectory analysis using Monocle3 (H) and Slingshot (I), both of which identify a differentiation path from HM toward the AM3 subtype. (J) Hcar2 expression plotted along the pseudotime trajectory, demonstrating progressive upregulation during differentiation. (K) Gene ontology (GO) enrichment analysis for marker genes of each microglial subtype.

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated overnight at 4°C with the indicated primary antibodies: against Hcar2 (1:2000; AWA45885 ; Abiowell), Arg‐1 (1:2000; sc‐271430; Santa Cruz Biotechnology), CD206 (1:2000; ET1702‐04; HUABIO), TGF‐β (1:2000; AWA10316 ; Abiowell), or HRP‐conjugated β‐actin (1:10000; 700068; Zenbio).

Techniques: Expressing, Marker

Journal: Clinical and Translational Medicine

Article Title: Niacin promotes motor function recovery after spinal cord injury via Hcar2‐dependent microglia immunometabolic regulation

doi: 10.1002/ctm2.70683

Figure Lengend Snippet: Hcar2 protein expression was upregulated in perilesional microglia following SCI. (A) Schematic timeline of the in vivo experimental design, indicating spinal cord tissue collection points (days post‐injury, dpi) and the definition of the IA and SA regions. (B) Representative Western blot analysis of Hcar2 protein levels in spinal cord lysates at 1, 3, 7 and 14 dpi. β‐Tubulin served as the loading control ( n = 4). (C) Densitometric quantification of Hcar2 protein levels relative to those of β‐Tubulin ( n = 4). (D) Representative confocal images of spinal cord sections at 7 dpi stained for Iba‐1 (microglia, grey) and Hcar2 (green). Nuclei were counterstained with DAPI (blue). Scale bars: 500 µm (overview), 100 µm (inset). (E) Fluorescence intensity line scan profile across a representative Iba‐1 + Hcar2 + cell in the IA region, demonstrating signal colocalization ( n = 5). (F) Quantification of the Hcar2 + area as a percentage of the total Iba‐1 + microglial area ( n = 5). (G) Representative immunofluorescence images of BV2 cells treated with vehicle or LPS (100 ng/mL) for 24 h and stained for Iba‐1 (red) and Hcar2 (green). Scale bar: 100 µm. (H) Quantification of Hcar2 immunofluorescence intensity in BV2 cells ( n = 3 independent experiments). (I) Representative Western blot analysis of Hcar2 protein levels in BV2 cell lysates. (J) Densitometric quantification of Hcar2 protein levels relative to those of β‐tubulin ( n = 3 independent experiments). The data are presented as the means ± SD. Statistical significance was determined via one‐way ANOVA with Tukey's post hoc test. Normal distribution was confirmed using the Shapiro–Wilk test. ** p < .01, *** p < .001 vs. Sham. ### p < .001 vs. SCI‐IA; ns, not significant.

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated overnight at 4°C with the indicated primary antibodies: against Hcar2 (1:2000; AWA45885 ; Abiowell), Arg‐1 (1:2000; sc‐271430; Santa Cruz Biotechnology), CD206 (1:2000; ET1702‐04; HUABIO), TGF‐β (1:2000; AWA10316 ; Abiowell), or HRP‐conjugated β‐actin (1:10000; 700068; Zenbio).

Techniques: Expressing, In Vivo, Western Blot, Control, Staining, Fluorescence, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: Niacin promotes motor function recovery after spinal cord injury via Hcar2‐dependent microglia immunometabolic regulation

doi: 10.1002/ctm2.70683

Figure Lengend Snippet: Hcar2 deficiency induced a metabolic shift toward oxidative phosphorylation and exacerbated neuroinflammation. (A) Schematic illustration of the Hcar2 knockout (KO) strategy. (B) qRT‒PCR confirmation of Hcar2 mRNA expression in the spinal cords of Hcar2 +/+ and Hcar2 −/− mice ( n = 6). (C) Schematic workflow of the in vivo study, including SCI modelling, tissue harvesting at 7 dpi, RNA sequencing, and qRT‒PCR validation. (D and E) KEGG (D) and Reactome (E) pathway enrichment analyses of differentially expressed genes (DEGs) between Hcar2 +/+ and Hcar2 −/− mice, highlighting the downregulation of metabolic pathways ( n = 3). (F–H) qRT‒PCR analysis of the expression of the microglial markers Arg1 (F), Cd206 (G), and Cd86 (H) in the spinal cord at 7 dpi ( n = 6). (I) Representative images of immunofluorescence staining for Iba‐1 (green) and Arg‐1 (red) in the lesion core from Hcar2 +/+ and Hcar2 −/− Hcar2 +/+ mice at 7 dpi. Scale bars: 500 µm (overview), 100 µm (inset) ( n = 4). (J, K) Quantification of Iba‐1 + Arg‐1 + cell density (J) and the percentage of Arg‐1 + cells within the Iba‐1 + population (K) ( n = 4). (L‐N) Multiplex flow cytometric analysis of IL‐6 (L), IL‐1β (M) and TNF‐α (N) in spinal cord lysates ( n = 4). (O) Representative Western blots of M2‐like markers (Arg‐1, CD206 and TGF‐β) ( n = 4). (P–R) Densitometric quantification of Arg‐1 (P), CD206 (Q) and TGF‐β (R) protein levels normalized to those of β‐actin ( n = 4). The data are presented as the means ± SD. Statistical significance was determined via one‐way ANOVA with Tukey's post hoc test. Normal distribution was confirmed using the Shapiro–Wilk test. * p < .05, ** p < .01, *** p < .001 vs. Hcar2 +/+ +Sham. # p < .05, ### p < .001 vs. Hcar2 +/+ +SCI; ns, not significant.

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated overnight at 4°C with the indicated primary antibodies: against Hcar2 (1:2000; AWA45885 ; Abiowell), Arg‐1 (1:2000; sc‐271430; Santa Cruz Biotechnology), CD206 (1:2000; ET1702‐04; HUABIO), TGF‐β (1:2000; AWA10316 ; Abiowell), or HRP‐conjugated β‐actin (1:10000; 700068; Zenbio).

Techniques: Phospho-proteomics, Knock-Out, Expressing, In Vivo, RNA Sequencing, Biomarker Discovery, Immunofluorescence, Staining, Multiplex Assay, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Niacin promotes motor function recovery after spinal cord injury via Hcar2‐dependent microglia immunometabolic regulation

doi: 10.1002/ctm2.70683

Figure Lengend Snippet: Niacin promoted an anti‐inflammatory phenotype in vitro through reprogramming microglial immunometabolism. (A) Schematic of the in vitro experimental design involving BV2 microglia challenged with LPS (100 ng/mL) and treated with niacin (NA). (B) Representative images of Iba‐1 (green) and Arg‐1 (red) immunofluorescence staining in BV2 cells treated with increasing concentrations of NA (0.1, 0.3 or 1 mM) for 24 h. Scale bar: 100 µm. (C) Quantification of OCR in BV2 cells, showing the NA‐mediated rescue of mitochondrial respiration. (D) Quantification of the red/green fluorescence intensity ratio from JC‐1 staining, which represents the relative mitochondrial membrane potential (ΔΨm). (E) Representative images of JC‐1 staining showing J‐aggregates (red, high potential) and J‐monomers (green, low potential). Scale bar: 100 µm. (F) Quantification of intracellular ATP levels. (G and H) qRT‒PCR analysis of the mRNA expression levels of the proinflammatory cytokines IL‐6 (G) and IL‐1β (H) normalized to that of β‐actin. (I) Schematic of the in vitro experimental design involving shRNA‐mediated Hcar2 knockdown in BV2 cells followed by LPS (100 ng/mL) and NA (0.3 mM) treatment. (J) qRT‒PCR confirmation of Hcar2 mRNA expression indicating knockdown efficiency. (K) Quantification of OCR in Hcar2 ‐knockdown BV2 cells. (L) Quantification of the red/green fluorescence intensity ratio from JC‐1 staining, representing the relative ΔΨm in Hcar2 ‐knockdown cells. (M) Representative images of JC‐1 staining in Hcar2 ‐knockdown BV2 cells showing J‐aggregates (red) and J‐monomers (green). Scale bar: 100 µm. (N) Quantification of intracellular ATP levels in Hcar2 ‐knockdown cells. (O and P) qRT‒PCR analysis of the mRNA expression levels of the proinflammatory cytokines IL‐6 (O) and IL‐1β (P) in Hcar2 ‐knockdown cells, normalized to that of β‐actin. Data are presented as the means ± SD. Statistical significance was determined via one‐way ANOVA with Tukey's post hoc test. Normal distribution was confirmed using the Shapiro–Wilk test. In A‐H, * p < .05, *** p < .001 vs. Control. # p < .05, ## p < .01, ### p < .001 vs. LPS; In I‐P, * p < .05, ** p < .01, *** p < .001 vs. NC+LPS. ### p < .001 vs. NC+LPS+NA; ns, not significant; n = 3 independent experiments.

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated overnight at 4°C with the indicated primary antibodies: against Hcar2 (1:2000; AWA45885 ; Abiowell), Arg‐1 (1:2000; sc‐271430; Santa Cruz Biotechnology), CD206 (1:2000; ET1702‐04; HUABIO), TGF‐β (1:2000; AWA10316 ; Abiowell), or HRP‐conjugated β‐actin (1:10000; 700068; Zenbio).

Techniques: In Vitro, Immunofluorescence, Staining, Fluorescence, Membrane, Expressing, shRNA, Knockdown, Control

Journal: Clinical and Translational Medicine

Article Title: Niacin promotes motor function recovery after spinal cord injury via Hcar2‐dependent microglia immunometabolic regulation

doi: 10.1002/ctm2.70683

Figure Lengend Snippet: Niacin promoted neuroprotection and locomotor recovery after SCI in a Hcar2‐dependent manner. (A) Experimental timeline for niacin (NA) administration and behavioural assessments. (B) Representative double immunofluorescence images of the perilesional spinal cord at 7 dpi showing Iba‐1 (microglia, red) and HK2 (glycolytic enzyme, green) expression. Scale bar: 100 µm. (C) Representative immunofluorescence images of the perilesional spinal cord at 28 dpi showing GFAP (astrocytes, green) and ChAT (motor neurons, red) expression. The high‐magnification inset displayed a representative region rostral to the lesion core, as indicated by the white box. Scale bars: 500 µm (overview), 100 µm (inset). (D) Quantification of the HK2 + area within the Iba‐1 + microglial population at 7 dpi ( n = 4). (E) Quantification of surviving ChAT + motor neurons in the ventral horn (n = 4). (F, G) CatWalk gait analysis at 28 dpi, displaying representative footprint patterns (F) and 3D intensity maps (G). (H–J) Quantification of key gait parameters: average run speed (H), mean intensity (I), and maximum contact mean intensity (J) of the hindlimbs ( n = 9). (K) Time course of locomotor recovery assessed by Basso Mouse Scale (BMS) scores over 28 days ( n = 9). The data are presented as the means ± SD. Statistical significance was determined via one‐way ANOVA (D‐E, H–J) or two‐way repeated‐measures ANOVA (K) followed by Tukey's post hoc test. Normal distribution was confirmed using the Shapiro–Wilk test. *** p < .001 vs. Sham. # p < .05, ## p < .01, ### p < .001 vs. SCI+NA; ns, not significant.

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated overnight at 4°C with the indicated primary antibodies: against Hcar2 (1:2000; AWA45885 ; Abiowell), Arg‐1 (1:2000; sc‐271430; Santa Cruz Biotechnology), CD206 (1:2000; ET1702‐04; HUABIO), TGF‐β (1:2000; AWA10316 ; Abiowell), or HRP‐conjugated β‐actin (1:10000; 700068; Zenbio).

Techniques: Immunofluorescence, Expressing

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