Journal: Journal of Biological Chemistry

Article Title: Nicotinic Acid Receptor Agonists Differentially Activate Downstream Effectors

doi: 10.1074/jbc.m701866200

Figure Lengend Snippet: FIGURE 1. Structures of GPR109A agonists presented herein with compound designations indicated below each structure.

Article Snippet: Measurement of Adenylyl Cyclase Inhibition A 96-well Adenylyl Cyclase Activation Flashplate AssayTM kit (PerkinElmer) was optimized to measure changes in intracellular cAMP levels due to receptor activation in CHO-K1 stable cell lines expressing GPR109A.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Nicotinic Acid Receptor Agonists Differentially Activate Downstream Effectors

doi: 10.1074/jbc.m701866200

Figure Lengend Snippet: FIGURE6.MouseflushingviaLaserDoppler.Base-lineperfusionwasestablishedfor3minintheventralright ear of C57Bl/6 mice. Mice were then given intraperitoneal injections of individual GPR109A agonists. Values are the mean % change from base-line, preinjection values S.E. of 3–26 individual animals as indicated in the key at right.

Article Snippet: Measurement of Adenylyl Cyclase Inhibition A 96-well Adenylyl Cyclase Activation Flashplate AssayTM kit (PerkinElmer) was optimized to measure changes in intracellular cAMP levels due to receptor activation in CHO-K1 stable cell lines expressing GPR109A.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Nicotinic Acid Receptor Agonists Differentially Activate Downstream Effectors

doi: 10.1074/jbc.m701866200

Figure Lengend Snippet: FIGURE 9. Qualitative assessment of GPR109A internalization following agonist exposure. The dose- dependent internalization of GPR109A was examined using immunofluorescence microscopy on tran- siently transfected COS-7 cells following 30 min of exposure to the indicated doses of nicotinic acid (left), compound 1a (middle), or compound 4a (right). Data shown are representative of at least three independ- ent experiments.

Article Snippet: Measurement of Adenylyl Cyclase Inhibition A 96-well Adenylyl Cyclase Activation Flashplate AssayTM kit (PerkinElmer) was optimized to measure changes in intracellular cAMP levels due to receptor activation in CHO-K1 stable cell lines expressing GPR109A.

Techniques: Immunofluorescence, Microscopy, Transfection

Journal: Journal of Biological Chemistry

Article Title: Nicotinic Acid Receptor Agonists Differentially Activate Downstream Effectors

doi: 10.1074/jbc.m701866200

Figure Lengend Snippet: FIGURE 10. Qualitative assessment of GPR109A internalization following 10 M agonist exposure for 30 min. Internalization of GPR109A was examined using immunofluorescence microscopy on transiently trans- fected COS-7 cells following 30 min of exposure to 10 M of indicated compounds. Data shown are represent- ative of at least three independent experiments.

Article Snippet: Measurement of Adenylyl Cyclase Inhibition A 96-well Adenylyl Cyclase Activation Flashplate AssayTM kit (PerkinElmer) was optimized to measure changes in intracellular cAMP levels due to receptor activation in CHO-K1 stable cell lines expressing GPR109A.

Techniques: Immunofluorescence, Microscopy

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: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: The relative expression of GPR109A, IL-6, TNF-α , and IL-1β . The mammary glands were collected from healthy dairy cows and mastitis dairy cows ( n = 6). ( a , b ) The results of hematoxylin and eosin (H&E) staining and immunohistochemistry assays in the control and mastitis dairy cows. ( c ) The gene levels of GPR109A in the control and mastitis dairy cows. ( d – f ) The expression of pro-inflammatory factors in the control and mastitis dairy cows. The mRNA levels of GPR109A ( g ), IL-6 ( d ), TNF-α ( e ), and IL-1β ( f ) were normalized to the level of β-actin . The values are presented as the means ± SD (∗ p < 0.05 and ∗∗ p < 0.01).

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: Expressing, Staining, Immunohistochemistry, Control

Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: Effects of autophagy, GPR109A, and NRF2 on BMECs inflammation. ( a – i ) The bovine mammary epithelial cells (BMECs) were pre-treated with niacin and NC shRNA, shRNA, retinoic acid (RA), or 3-methyladenine (3-MA) for 1 h and then stimulated with LPS for 24 h. The mRNA levels of IL-6, IL-1β , and TNF-α were determined by qRT-PCR. The mRNA levels of IL-6, IL-1β , and TNF-α were normalized to the level of β-actin . The values are presented as the means ± SD (∗ p < 0.05 and ∗∗ p < 0.01).

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: shRNA, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: Niacin can activate the GPR109A/NRF2/autophagy signal pathway. The cells were collected at 0, 3, 6, 12, and 24 h to extract the total protein. The total protein was prepared and subjected to Western blotting using GPR109A, NRF-2, HO-1, and β-tubulin antibodies. T-NRF-2 means total NRF-2. ( a – d ) The protein levels of GPR109A, NRF-2, and HO-1. The cells from different experimental groups were treated with niacin or shRNA+niacin for 24 h, and then, the total protein was collected. N-NRF-2 means NRF-2 in the nucleus. C-NRF-2 means NRF-2 in the cytoplasm. ( e – h ) The protein levels of GPR109A, C-NRF-2, N-NRF-2, and HO-1. Each immunoreactive band was digitized and expressed as a ratio of the β-tubulin level. ( i ) The immunofluorescence results of the assay for NRF-2. The scale length in the figure is 200 μM. ( j ) The relative fluorescence intensity of NRF-2/ARE. The mRNA levels were determined by qRT-PCR. ( k ) The mRNA levels of ATG12, ATG4D, p62, ATG5, ULK1, ATG4B, Beclin, LC3B , and ATG7 were normalized to the level of β-actin . The values are presented as the means ± SD (∗ p < 0.05 and ∗∗ p < 0.01).

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: Western Blot, shRNA, Immunofluorescence, Fluorescence, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: GPR109A regulates energy metabolism, AMPK phosphorylation, and P62 interact with Keap-1 in BMECs. The cells were collected at 0, 3, 6, 12, and 24 h to extract the total protein. ( a , b ) The protein levels of p-AMPK. The cells from different experimental groups were treated with niacin or shRNA + niacin for 24 h, and then, the total protein was collected. The cell lysates were prepared and subjected to Western blotting using AMPK and p-AMPK antibodies. ( c , d ) The protein levels of p-AMPK. Each immunoreactive band was digitized and expressed as a ratio of the β-tubulin. ( e ) The BMECs were pre-treated with niacin, niacin+shRNA, or shRNA for 24 h. The cells were then collected, and the ATP, ADP, and AMP levels were detected by liquid chromatography ( n = 3). ( f – h ) of the ATP, ADP, and AMP content in the BMECs after adding niacin, niacin + shRNA or shRNA. ( i ) The ratio of (AMP + ADP)/ATP. The values are presented as the means ± SD (ns means no difference, ∗ p < 0.05 and ∗∗ p < 0.01).

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: Phospho-proteomics, shRNA, Western Blot, Liquid Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: GPR109A alleviates inflammation of BMECs by regulating autophagy. Cells from different experimental groups were treated with niacin, LPS, or niacin+LPS for 24 h, and then, the total protein was collected. The cell lysates were prepared and subjected to Western blotting using β-tubulin ( a ), LC3B ( a , b ), and P62 ( a , c ) antibodies. Each immunoreactive band was digitized and expressed as a ratio of the β-tubulin. ( d ) The left panel shows 6000 × 120 V; the right panel shows is 12,000 × 120 V. The results revealed by the electron microscopy showed that niacin could induce autophagy in the non-treated BMECs and LPS-treated BMECs. ( e – m ) The mRNA levels were determined by qRT-PCR. The mRNA levels of ATG12, ATG4D , p62 , ATG5, ULK1, ATG4B, Beclin, LC3B , and ATG7 were normalized to the level of β-actin . Values are presented as the means ± SD (∗ p < 0.05 and ∗∗ p < 0.001).

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: Western Blot, Electron Microscopy, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: Effect of GPR109A on the AMPK/NRF-2/HO-1, P38, JNK1/2, and ERK1/2 signaling pathways. The cells from different experimental groups were treated with niacin, niacin, or LPS for 24 h, and then, the total protein was collected. N-P65 indicates P65 in the nucleus. N-NRF-2 means NRF-2 in the nucleus. The cell lysates were prepared and subjected to Western blotting using antibodies for P38 ( a , d ), p-P38 ( a , d ), JNK1/2 ( a , b ), p-JNK1/2 ( a , b ), ERK1/2 ( a , c ), p-ERK1/2 ( a , c ), AMPK ( a , f ), p-AMPK ( a , f ), HO-1 ( a , g ), β-tubulin ( e ), N-P65 ( a , h ), N-NRF-2 ( a , i – k ), and laminin B ( e ).

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: Protein-Protein interactions, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: The primer sequenceof the genes.

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: Sequencing

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: Cell death and differentiation

Article Title: Neutrophil apoptosis mediated by nicotinic acid receptors (GPR109A).

doi: 10.1038/sj.cdd.4402238

Figure Lengend Snippet: Figure 1 Mature blood neutrophils, but not immature bone marrow neutrophils or mature blood eosinophils, express the NA receptor GPR109A on their surface. (a) Flow cytometry. Cells were stained with control (black) or anti-GPR109A (green) mAbs. (b and c) Confocal microscopy. Neutrophils were stained with FITC- conjugated NA and anti-GPR109A mAb, respectively. Nuclei were stained with PE in (b). Results in each panel are representative of at least four independent experiments

Article Snippet: Anti-GPR109A (HM74a) rat mAb was from R&D Systems Europe Ltd. (Abingdon, UK).

Techniques: Flow Cytometry, Staining, Control, Confocal Microscopy

Journal: Renal Failure

Article Title: Oral Astragalus polysaccharide alleviates adenine-induced kidney injury by regulating gut microbiota–short-chain fatty acids–kidney G protein-coupled receptors axis

doi: 10.1080/0886022x.2024.2429693

Figure Lengend Snippet: Figure 6. APS supplementing increase kidney GRP expression. (A) Kidney GPR41 immunohistochemical staining and analysis (×100 and ×200). (B) Kidney GPR43 immunohistochemical staining and analysis (×100 and ×200). (C) Kidney GPR109a immunohistochemical staining and analysis (×100 and ×200). (D) Western-blot analysis of GPR41, 43, and 109a in kidney tissue. The data are presented as the mean ± standard deviation of the mean for each mea surement (*p < .05, **p < .01).

Article Snippet: Sections were incubated with anti-G protein-coupled receptor 41 (GPR41) primary antibody (1:100, Lot: 2440.Pb1.AP, NOVUS, Centennial, CO), anti-GPR43 primary antibody (1:50, Lot: #617A024, Absin, Shanghai, China), and anti-GPR109a primary antibody (1:100, Lot: E105361, NOVUS, Centennial, CO) at 4 °C overnight.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Standard Deviation