Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 1. Identification and charac- terization of GPCR135 ligand activity in rat brain extracts. A, GPCR135 li- gand activity in different rat tissues. Ex- tracted rat tissues at the dilutions shown were used as ligands in the GTPS bind- ing assay using human GPCR135 ex- pressing cell membrane. B, molecular weight characterization of GPCR135 li- gand from rat brain extract. Crude rat brain ethanol/HCl extract was run through a HPLC gel filtration column. Fractions were collected and assayed for GPCR135 ligand activity in GTPS bind- ing assays using human GPCR135 ex- pressing cell membranes. In a parallel ex- periment, peptides and nucleotides with known molecular weights were run using the same conditions to serve as the molec- ular mass standards.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Activity Assay, Membrane, Molecular Weight, Filtration

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 2. Purification and characterization of porcine GPCR135 ligand. After extraction, fractionation, two rounds of ion exchange, and two rounds of reverse phase chromatographies, homogeneous peptide was obtained. A, the final RP-HPLC. Fractions 39–40 showed ligand activity for GPCR135 as demonstrated in GTPS binding assays. B, GTPS binding assays for GPCR135 ligand activity in the RP-HPLC fractions. C, the porcine GPCR135 ligand in fraction 39-40 was subjected to amino acid sequence analysis. The resulting amino acid sequences are aligned to human, mouse, and rat relaxin-3. All cysteine residues are highlighted in bold letters. The three amino acids in porcine GPCR135 ligand that differ from human relaxin-3 are underlined.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Purification, Extraction, Fractionation, Activity Assay, Binding Assay, Sequencing

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 3. Expression, purification, and characterization of recombinant human relaxin-3. A, schematic diagram of the predicted amino acid sequence for the human relaxin-3 expression construct, relaxin-3RR. Arrows indicate an enterokinase cleavage site (DDDDK) and two furin cleavage sites (RWRR and RGRR). The amino acid R with the star (*) indicates the mutation made from the natural sequence of RGSR to RGRR, an artificial furin site in the construct. The numbers indicate the corresponding positions of the amino acid in the natural human relaxin-3 pre-propeptide. B, RP-HPLC analysis of purified recombinant human relaxin-3. The purified human relaxin-3 was run in a C-18 RP-HPLC analytical column in 0.1% trifluoroacetic acid with an acetonitrile (ACN) gradient. C, fractions from B were assayed for GPCR135 ligand activity in GTPS binding assays using GPCR135 expressing cell membranes.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Expressing, Purification, Recombinant, Sequencing, Construct, Mutagenesis, Activity Assay, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 4. Characterization of GPCR135 using 125I-relaxin-3 as the radioligand. A, saturation isotherm binding for 125I-relaxin-3 and GPCR135. GPCR135 expressing cell membranes were used in binding assays with different concentrations of 125I-relaxin-3 as the radioligand. Nonspecific binding was determined using the same conditions in the presence of 5 M unlabeled relaxin-3. B, competition binding analysis of GPCR135. GPCR135 expressing cell membranes were used to perform radioligand binding assays with 100 pM 125I-relaxin-3 as the radioligand. Different unlabeled peptides at various concentrations were used as competitors.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Binding Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 5. Functional characterization of GPCR135 using relaxin-3 and related peptides as ligands. A, relaxin-3 stimulates 35S-GTPS binding in GPCR135 expressing cells. Different peptides were added at various concentrations to the human GPCR135 expressing cell membranes to stimulate GTPS incorporation. The specific 35S-GTPS incorporation was obtained by subtracting counts without ligand from the counts with ligand. B, inhibition of forskolin-stimulated cAMP production by relaxin-3. Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences). C, dose response of relaxin-3 inhibition of cAMP production in GPCR135 expressing cells. Chinese hamster ovary cells stably expressing GPCR135 were stimulated with different concentrations of peptides, including relaxin-3, at various concentrations. Forskolin was then added to all samples at a final concentration of 5 M. cAMP from the stimulated cells was extracted and measured using cAMP flash plates. D, relaxin-3 stimulates Ca2 mobilization in HEK293 cells co-expressing GPCR135 and Gqi5. HEK293 cells, either mock transfected (293), transfected with Gqi5 (293/Gqi5), human GPCR135 (GPCR135), or co-transfected with human GPCR135 and Gqi5 (GPCR135/Gqi5), were used for Ca2 mobilization assays. Relaxin-3 stimulated intracellular Ca2 mobilization was monitored by FLIPR.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Functional Assay, Binding Assay, Expressing, Inhibition, Stable Transfection, Control, Concentration Assay, Transfection

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 6. A, RT-PCR detection of GPCR135 and relaxin-3 mRNA expression profiles in different human tissues. The PCR products were run in 2% agarose gels, stained with ethidium bromide, and visualized under UV irradiation. In parallel, PCRs amplifying human -actin cDNA served as the controls. B–E, coronal brain sections showing expression of GPCR135 and relaxin-3 mRNA by in situ hybridization. B, autoradiogram shows that GPCR135 mRNA distribution is distinct in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). C, brightfield photomicrograph of the paraventricular nucleus showing expression of GPCR135 mRNA. Strong hybridization signal is seen as dark grains. Magnification 20. D, relaxin-3 mRNA distribution in the central gray and nucleus incertus. E, brightfield photomicrograph of central gray (CG) and nucleus incertus (NI) showing expression of relaxin-3 mRNA. Magnification 2. B and D in the figure are pseudocolor images from autoradiograms processed on the Fuji Film Bio-Imaging Analyzer System. Colors represent relative levels of hybridization densities with the rank order of red yellow green blue black.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Irradiation, In Situ Hybridization, Hybridization, Imaging

Journal: BioMed Research International

Article Title: RLN2 Is a Positive Regulator of AKT-2-Induced Gene Expression Required for Osteosarcoma Cells Invasion and Chemoresistance

doi: 10.1155/2015/147468

Figure Lengend Snippet: Western blot assay for RLN2, RXFP1, AKT, ERK1/2, p-Akt, and p-ERK1/2 in OS tissues with pulmonary metastatic disease.

Article Snippet: Antibodies to RLN2, LGR7, p-Akt (Ser473), p-ERK1/2 (Phospho-Thr202/Tyr204), AKT, ERK1/2, NF- κ B (p65), MMP-9, VEGF, Bcl-2, GAPDH, RLN2 siRNA (h), and recombinant human RLN2 (B-29/A-24) were from Santa Cruz Biotech Inc. (USA).

Techniques: Western Blot

Journal: BioMed Research International

Article Title: RLN2 Is a Positive Regulator of AKT-2-Induced Gene Expression Required for Osteosarcoma Cells Invasion and Chemoresistance

doi: 10.1155/2015/147468

Figure Lengend Snippet: Expression of RLN2 in OS cells following different treatment. (a) The expression of RLN2 protein was measured by western blot in MG-63 cells with specific siRNA transfection. The result showed that RLN2 was significantly blocked in positive groups compared with control group. (b) U-2OS cells were treated with 100 nM recombinant relaxin for 24 hs. The expression of RLN2 protein was measured by western blot in MG-63 cells. The result showed that RLN2 was significantly increased in positive groups compared with control group. ∗ P < 0.05; ∗∗ P < 0.01, versus control.

Article Snippet: Antibodies to RLN2, LGR7, p-Akt (Ser473), p-ERK1/2 (Phospho-Thr202/Tyr204), AKT, ERK1/2, NF- κ B (p65), MMP-9, VEGF, Bcl-2, GAPDH, RLN2 siRNA (h), and recombinant human RLN2 (B-29/A-24) were from Santa Cruz Biotech Inc. (USA).

Techniques: Expressing, Western Blot, Transfection, Control, Recombinant

Journal: BioMed Research International

Article Title: RLN2 Is a Positive Regulator of AKT-2-Induced Gene Expression Required for Osteosarcoma Cells Invasion and Chemoresistance

doi: 10.1155/2015/147468

Figure Lengend Snippet: Effect of RLN2 inhibition decreased AKT/NF- κ B signaling pathway in MG-63 cells. MG-63 cells were transfected with RLN2 siRNA2 for 48 hs and then treated with myr-AKT or TNF- α at different time points. (a) The protein of p-AKT (Ser473), p-ERK1/2, NF- κ B (p65), MMP-9, VEGF, and bcl-2 was measured using western blot analysis. (b) Nuclear extracts were prepared and subjected to analysis for NF- κ B DNA-binding activity as measured by EMSA. Retinoblastoma protein level served as nuclear protein loading control. (c) Zymographic analysis of MMP-9 gelatinolytic activity, which was quantified by densitometry and graphed in OD units (mean ± SEM) ( ∗ P < 0.05).

Article Snippet: Antibodies to RLN2, LGR7, p-Akt (Ser473), p-ERK1/2 (Phospho-Thr202/Tyr204), AKT, ERK1/2, NF- κ B (p65), MMP-9, VEGF, Bcl-2, GAPDH, RLN2 siRNA (h), and recombinant human RLN2 (B-29/A-24) were from Santa Cruz Biotech Inc. (USA).

Techniques: Inhibition, Transfection, Western Blot, Binding Assay, Activity Assay, Control

Journal: BioMed Research International

Article Title: RLN2 Is a Positive Regulator of AKT-2-Induced Gene Expression Required for Osteosarcoma Cells Invasion and Chemoresistance

doi: 10.1155/2015/147468

Figure Lengend Snippet: Effect of RLN2 overexpression increased AKT/NF- κ B signaling pathway in U-2OS cells. (a) U-2OS cells were treated with different agents at different time points. The protein of p-AKT (Ser473), p-ERK1/2, NF- κ B (p65), MMP-9, VEGF, and bcl-2 was measured using western blot analysis. (b) U-2OS cells were treated with different agents at different time points. Nuclear proteins were extracted by freeze-thaw lysis using buffer C in U-2OS cells for electrophoretic mobility shift assays (EMSA). Retinoblastoma protein level served as nuclear protein loading control. (c) U-2OS cells were treated with different agents at different time points. Zymographic analysis of MMP-9 gelatinolytic activity, which was quantified by densitometry and graphed in OD units (mean ± SEM) ( ∗ P < 0.05).

Article Snippet: Antibodies to RLN2, LGR7, p-Akt (Ser473), p-ERK1/2 (Phospho-Thr202/Tyr204), AKT, ERK1/2, NF- κ B (p65), MMP-9, VEGF, Bcl-2, GAPDH, RLN2 siRNA (h), and recombinant human RLN2 (B-29/A-24) were from Santa Cruz Biotech Inc. (USA).

Techniques: Over Expression, Western Blot, Lysis, Electrophoretic Mobility Shift Assay, Control, Activity Assay

Journal: BioMed Research International

Article Title: RLN2 Is a Positive Regulator of AKT-2-Induced Gene Expression Required for Osteosarcoma Cells Invasion and Chemoresistance

doi: 10.1155/2015/147468

Figure Lengend Snippet: Effect of RLN2 on cell growth and apoptosis. (a) RLN2 siRNA2 was transfected into the MG-63 cell for 1–5 days; cell survival rate was performed with MTT assay. (b) Cell apoptosis was detected by Annexin V-FITC/PI staining method. (c) U-2OS cells were treated with recombinant relaxin for 5 days, and then we performed determination of cell survival rate with MTT assay. ∗ P < 0.05, versus control.

Article Snippet: Antibodies to RLN2, LGR7, p-Akt (Ser473), p-ERK1/2 (Phospho-Thr202/Tyr204), AKT, ERK1/2, NF- κ B (p65), MMP-9, VEGF, Bcl-2, GAPDH, RLN2 siRNA (h), and recombinant human RLN2 (B-29/A-24) were from Santa Cruz Biotech Inc. (USA).

Techniques: Transfection, MTT Assay, Staining, Recombinant, Control

Journal: BioMed Research International

Article Title: RLN2 Is a Positive Regulator of AKT-2-Induced Gene Expression Required for Osteosarcoma Cells Invasion and Chemoresistance

doi: 10.1155/2015/147468

Figure Lengend Snippet: RLN2 regulates sensitivity of OS cells to cisplatin. (a) MG-63 cells were transfected with RLN2 siRNA2 and then treated with myr-AKT (10 μ M) for 24 hs or treated with 20 ng/mL of TNF- α for 6 hs, then following 10 μ g/mL cisplatin treatment for 48 hs. (b) U-2OS cells were treated with NF- κ B inhibitor BAY 11-7082 (10 μ M) for 24 hs, or 50 μ M LY294002 for 3 hs, or I κ B α M (100 nM) for 24 hs; then, the cells were treated with recombinant relaxin for 24 hs, after which the cells were treated with 10 μ g/mL cisplatin for 48 hs. Cell apoptosis was detected by Annexin V-FITC/PI staining method. The experiments were done in triplicate for each sample, and analyses were performed using a FACScan flow cytometer ( ∗ P < 0.05).

Article Snippet: Antibodies to RLN2, LGR7, p-Akt (Ser473), p-ERK1/2 (Phospho-Thr202/Tyr204), AKT, ERK1/2, NF- κ B (p65), MMP-9, VEGF, Bcl-2, GAPDH, RLN2 siRNA (h), and recombinant human RLN2 (B-29/A-24) were from Santa Cruz Biotech Inc. (USA).

Techniques: Transfection, Recombinant, Staining, Flow Cytometry

Journal: BioMed Research International

Article Title: RLN2 Is a Positive Regulator of AKT-2-Induced Gene Expression Required for Osteosarcoma Cells Invasion and Chemoresistance

doi: 10.1155/2015/147468

Figure Lengend Snippet: Effect of RLN2 on invasion and angiogenesis in OS cells. MG-63 cells were transfected with RLN2 siRNA2 for 48 hs and then transfected with myr-AKT (10 μ M) for 24 hs or treated with 20 ng/mL of TNF- α for 6 hs; the invasive ability of MG-63 cells was detected by Matrigel invasion assay (a). Quantitative analysis of the tube formation by HUVECs induced by conditioned medium (b). The reduced invasion and tube formation induced by the conditioned medium of RLN2-siRNA2 transfected MG-63 cells were rescued by adding myr-AKT or TNF- α . U-2OS cells were treated with NF- κ B inhibitor BAY 11-7082 (10 μ M) for 24 hs, or 50 μ M LY294002 for 3 hs, or I κ B α M (100 nM) for 24 hs; then, the cells were treated with recombinant relaxin for 24 hs; the invasive ability of U-2OS cells was detected by Matrigel invasion assay (c). Quantitative analysis of the tube formation by HUVECs induced by conditioned medium (d). The increased invasion and tube formation induced by the conditioned medium of RLN2 treated U-2OS cells were rescued by adding BAY 11-7082 or LY294002 or I κ B α M. ∗ P < 0.05.

Article Snippet: Antibodies to RLN2, LGR7, p-Akt (Ser473), p-ERK1/2 (Phospho-Thr202/Tyr204), AKT, ERK1/2, NF- κ B (p65), MMP-9, VEGF, Bcl-2, GAPDH, RLN2 siRNA (h), and recombinant human RLN2 (B-29/A-24) were from Santa Cruz Biotech Inc. (USA).

Techniques: Transfection, Invasion Assay, Recombinant

Journal: Bone Reports

Article Title: Relaxin 2 carried by magnetically directed liposomes accelerates rat midpalatal suture expansion and subsequent new bone formation

doi: 10.1016/j.bonr.2019.100202

Figure Lengend Snippet: Experimental protocol of palatal expansion and application of RLN2. (A) Schematic illustration of the experimental time course. Injection of liposome or RLN2-liposome into the palatal mucosa just over the midpalatal suture (MPS) was performed every 48 h (closed arrowheads). (B) Representative photograph of the MPS expansion fixed on the maxillary incisors of the rat, and micro-CT image of the rat maxillary area with the expansion appliance (E) and magnetic sheet (M). Closed arrowheads indicate injection points at intervals of 1.0 mm. (C) Body weights of rats in each group during the expansion and retention periods.

Article Snippet: Bilayer cholesterol liposomes (diameter < 100 nm) containing nanometer-sized magnetite (ferric oxide) particles for magnetic control of liposome accumulation and the fluorescent dye Cy5.5 for experimental in vivo imaging were used to localize recombinant human RLN2 protein (R&D Systems, Minneapolis, MN, USA) to the rat MPS.

Techniques: Injection, Micro-CT

Journal: Bone Reports

Article Title: Relaxin 2 carried by magnetically directed liposomes accelerates rat midpalatal suture expansion and subsequent new bone formation

doi: 10.1016/j.bonr.2019.100202

Figure Lengend Snippet: In vivo imaging of administered liposomes and IHC of RLN2, RXFP1, and RXFP2. (A) In vivo imaging of fluorescent Cy5.5 around the rat MPS by a preliminary examination. Warmer colors represent higher radiant efficiency. (B) IHC analysis of RLN2, RXFP1, and RXFP2 in the different groups. Arrows indicate Rxfp2-positive osteoblasts. Scale bar, 50 μm.

Article Snippet: Bilayer cholesterol liposomes (diameter < 100 nm) containing nanometer-sized magnetite (ferric oxide) particles for magnetic control of liposome accumulation and the fluorescent dye Cy5.5 for experimental in vivo imaging were used to localize recombinant human RLN2 protein (R&D Systems, Minneapolis, MN, USA) to the rat MPS.

Techniques: In Vivo Imaging, Liposomes

Journal: Journal of Infectious Diseases & Therapy

Article Title: A Deletion of Signal Peptide in Recombinant Murine Gamma-herpesvirus 68 M3 Protein Enhances its Binding Affinity to CCL5 Chemokine and Increases its Yield from Insect Cells

doi: 10.4172/2332-0877.1000347

Figure Lengend Snippet: Figure 1: Anti-M3 activities of mouse polyclonal antibody (A) and a set of newly prepared monoclonal antibodies (B, C) detected by indirect ELISA (A, B) and Western blot (C). (A) poly anti-M3-Ab/1 diluted 102 to 106 times with different amounts of purified recombinant MHV-68 M3 protein [40]; (B) media of a panel of twelve hybridoma cell lines (activities determined as an average of triplicates) and crude medium used as a negative control; (C a,b) specificity of anti-M3 antibody 1/27 (Ca) and poly anti-M3-Ab/1 (Cb) tested on MHV-68 infected BHK-21 cells: Lanes 1, 2 crude medium and cell lysate of uninfected cells; 3-4, 5-6, and 7-8 lysate of cells infected with MOI=2, 1, and 0.1; M-DNA molecular weight marker.

Article Snippet: Determination of the chemokine binding properties of M3del and M3 The binding of purified recombinant M3 proteins to the chemokines CCL5 and CXCL8 was determined using the Human CCL5/RANTES and Human CXCL8/IL-8 DuoSet ELISA kits (R&D Systems).

Techniques: Bioprocessing, Indirect ELISA, Western Blot, Purification, Recombinant, Negative Control, Infection, Molecular Weight, Marker

Journal: Journal of Infectious Diseases & Therapy

Article Title: A Deletion of Signal Peptide in Recombinant Murine Gamma-herpesvirus 68 M3 Protein Enhances its Binding Affinity to CCL5 Chemokine and Increases its Yield from Insect Cells

doi: 10.4172/2332-0877.1000347

Figure Lengend Snippet: Figure 2: Purification and specificity of recombinant MHV-68 M3 proteins prepared in insect Sf9 cells: M3del protein in baculovirus infected cell medium detected by SDS-PAGE (A) and Western blot using anti-M3 antibody 1/27 (B); affinity chromatography purified M3del detected by SDS-PAGE (C) and purified full-length M3 protein detected by Western blot (D). Lanes (A,B) 1 uninfected cells (negative control); 2-5 medium of infected cells at 1, 3, 6, 9 d.p.i.; 6 uninfected cells at 9 d.p.i.; M protein size marker; (C) 1 crude medium of infected cells at 6 d.p.i.; 2 flow-through fraction, non- binding proteins; 3 proteins washed away from the column with buffer A; 4-9 elution fractions E1-E6 eluted with buffer B; M- protein size marker; (D) 1-4 elution fractions E4, E3, E2 and E1 eluted with buffer B; 5 proteins washed away from the column with buffer A; 6 flow-through fraction, non-binding proteins; 7 crude medium of infected cells at 6 d.p.i.. The arrows indicate the M3 protein (~43 kDa).

Article Snippet: Determination of the chemokine binding properties of M3del and M3 The binding of purified recombinant M3 proteins to the chemokines CCL5 and CXCL8 was determined using the Human CCL5/RANTES and Human CXCL8/IL-8 DuoSet ELISA kits (R&D Systems).

Techniques: Purification, Recombinant, Infection, SDS Page, Western Blot, Affinity Chromatography, Negative Control, Marker, Binding Assay