Journal: The Journal of Biological Chemistry

Article Title: Competitive inhibition of the classical complement pathway using exogenous single-chain C1q recognition proteins

doi: 10.1016/j.jbc.2022.102113

Figure Lengend Snippet: Investigation of the binding properties of single-cell gC1q (sc-gC1q) variants by ELISA . Human and mouse IgG proteins (hIgG and mIgG, respectively) and human IgM were immobilized and titrated with each form of sc-gC1q variants ( A , B , and E ). In order to use the same primary antibody in assays examining neuronal pentraxins, we coated the wells with NPTX1 or NPTX2 and titrated with the different forms of sc-gC1q ( C and D ; marked with different colors). Hill equations ( lines ) were fitted to the data ( circles ) using Origin8 software. Values represent mean (from three replicates) ± SEM. gC1q, globular part of C1q; IgG, immunoglobulin G; IgM, immunoglobulin M; NPTX, neuronal pentraxin.

Article Snippet: Maxisorp 96-well ELISA plate (Nunc, Invitrogen) was coated with either 40 nM NPTX1 (human NPTX1; UniProtID: Q15818 ; catalog no.: 7707-NP-050; R&D Systems), NPTX2 (human NPTX2; UniProtID: P47972 ; catalog no.: 7816-NP-050; R&D Systems), sc-gC1q, sc-gC1q2, or sc-gC1q2l in PBS overnight at 4 °C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Competitive inhibition of the classical complement pathway using exogenous single-chain C1q recognition proteins

doi: 10.1016/j.jbc.2022.102113

Figure Lengend Snippet: SPR-binding studies . After covalent immobilization of IgG, IgM, NPTX1, and NPTX2) via amine coupling, the ligand channels were covalently blocked with bovine serum albumin (BSA) along with an empty channel that served as negative control. The sensorgrams ( pale lines ) are generated with the subtraction of the responses detected on the BSA channel from those detected on specific ligand channels. Dark lines represent the data fitting with a single exponential (sc-gC1q2 and sc-gC1q2l) or a double exponential (sc-gC1q). A – D , sc-gC1q analyte interactions with IgG, IgM, NPTX1, and NPTX2 ligands, respectively. E – H , sc-gC1q2 analyte interactions with IgG, IgM, NPTX1, and NPTX2 ligands, respectively. I – L , sc-gC1q2l analyte interactions with IgG, IgM, NPTX1, and NPTX2 ligands, respectively. gC1q, globular part of C1q; IgG, immunoglobulin G; IgM, immunoglobulin M; NPTX, neuronal pentraxin; sc-gC1q, single-chain gC1q; SPR, surface plasmon resonance.

Article Snippet: Maxisorp 96-well ELISA plate (Nunc, Invitrogen) was coated with either 40 nM NPTX1 (human NPTX1; UniProtID: Q15818 ; catalog no.: 7707-NP-050; R&D Systems), NPTX2 (human NPTX2; UniProtID: P47972 ; catalog no.: 7816-NP-050; R&D Systems), sc-gC1q, sc-gC1q2, or sc-gC1q2l in PBS overnight at 4 °C.

Techniques: Binding Assay, Negative Control, Generated, SPR Assay

Journal: The Journal of Biological Chemistry

Article Title: Competitive inhibition of the classical complement pathway using exogenous single-chain C1q recognition proteins

doi: 10.1016/j.jbc.2022.102113

Figure Lengend Snippet: Kinetic binding parameters of sc-gC1q variants measured by SPR a

Article Snippet: Maxisorp 96-well ELISA plate (Nunc, Invitrogen) was coated with either 40 nM NPTX1 (human NPTX1; UniProtID: Q15818 ; catalog no.: 7707-NP-050; R&D Systems), NPTX2 (human NPTX2; UniProtID: P47972 ; catalog no.: 7816-NP-050; R&D Systems), sc-gC1q, sc-gC1q2, or sc-gC1q2l in PBS overnight at 4 °C.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Competitive inhibition of the classical complement pathway using exogenous single-chain C1q recognition proteins

doi: 10.1016/j.jbc.2022.102113

Figure Lengend Snippet: Half inhibition values obtained from C4 ELISA

Article Snippet: Maxisorp 96-well ELISA plate (Nunc, Invitrogen) was coated with either 40 nM NPTX1 (human NPTX1; UniProtID: Q15818 ; catalog no.: 7707-NP-050; R&D Systems), NPTX2 (human NPTX2; UniProtID: P47972 ; catalog no.: 7816-NP-050; R&D Systems), sc-gC1q, sc-gC1q2, or sc-gC1q2l in PBS overnight at 4 °C.

Techniques: Inhibition

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Neuronal and Peripheral Pentraxins Modify Glutamate Release and may Interact in Blood-Brain Barrier Failure.

doi: 10.1093/cercor/bhx046

Figure Lengend Snippet: Figure 3. SAP forms complexes with each of the NPTXs and affects synaptic transmission. (A,B) Effect of coapplication of NPTX1 and SAP on paired-pulse

Article Snippet: Purified Native and Recombinant Pentraxin Proteins Recombinant human NPTXs were obtained from R&D Systems and human native purified SAP from Abcam.

Techniques: Transmission Assay

Journal: Scientific Reports

Article Title: NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

doi: 10.1038/srep36210

Figure Lengend Snippet: ( A ) RT-qPCR was performed during osteoblast differentiation using primers specific for ( A ) NPNT, ( B ) ALP, ( C ) Col1A1, and ( D ) OCN. Gene expression was normalized to 18S and compared to the expression at day 0. ( E ) NPNT expression construct (pcDNA3.1-NPNT-c-myc/His), which encodes a mouse full length NPNT, was generated. ( F ) Detection of NPNT protein in the conditioned medium of COS-7 cells transfected with NPNT expression construct using anti-c-myc antibody. ( G ) NPNT was detected in osteoblast supernatants using anti-NPNT antibody. HtrA1 was used as positive marker for osteoblast differentiation and β-Actin was used as loading control. Western blot images are presented as cropped format. Full length blots are presented in . *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant mouse NPNT (R&D Systems), human bFGF (PeproTech, Inc.), human VEGF (PeproTech, Inc.) and ERK1/2 inhibitor U0126 (Promega) were purchased for the assays.

Techniques: Quantitative RT-PCR, Gene Expression, Expressing, Construct, Generated, Transfection, Marker, Control, Western Blot

Journal: Scientific Reports

Article Title: NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

doi: 10.1038/srep36210

Figure Lengend Snippet: ( A ) Tibias from sham group (left) and OVX group (right) were visualized by μCT scanning, and the ROI focused on the trabecular region below the growth plate for quantitative analysis of ( B ) Tb.BV/TV, ( C ) Tb.Th, ( D ) Tb.N and ( E ) Tb.Sp. Real-time PCR quantitative analysis of ( F ) Runx2, ( G ) OCN and ( H ) NPNT gene expression in mouse forelimbs. n = 6/group. ( I ) μCT images of trabecular bone in the region between head and neck of femur from patients with osteoarthritis (top) and osteoporosis (bottom), and quantitative analysis of ( J ) Tb.BV/TV, ( K ) Tb.Th, ( L ) Tb.N and ( M ) Tb.Sp. Real-time PCR quantitative analysis of ( N ) Runx2, ( O ) OCN and ( P ) NPNT gene expression in human femora. n = 20/group. The gene expressions were normalized to β-Actin and 18S. *P < 0.05.

Article Snippet: Recombinant mouse NPNT (R&D Systems), human bFGF (PeproTech, Inc.), human VEGF (PeproTech, Inc.) and ERK1/2 inhibitor U0126 (Promega) were purchased for the assays.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression

Journal: Scientific Reports

Article Title: NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

doi: 10.1038/srep36210

Figure Lengend Snippet: ( A ) Representative microscopic images of H&E and immunostaining using NPNT and CD31-specific antibody on tibia sections of sham and OVX mice. Quantitative analysis of ( B ) NPNT and ( C ) CD31 protein expressions in the tibias of OVX mice. *P < 0.05, **P < 0.01.

Article Snippet: Recombinant mouse NPNT (R&D Systems), human bFGF (PeproTech, Inc.), human VEGF (PeproTech, Inc.) and ERK1/2 inhibitor U0126 (Promega) were purchased for the assays.

Techniques: Immunostaining

Journal: Scientific Reports

Article Title: NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

doi: 10.1038/srep36210

Figure Lengend Snippet: ( A ) Representative microscopic views of scratch wound healing assays performed using SVEC cells treated with recombinant mouse NPNT (500 ng/ml) from 0 to 16 hours. Scale bar, 100 μm. ( B ) Quantitative analysis of cell migration area. ( C ) Representative images showing tube-like structure formation by SVEC cells following treatment with recombinant mouse NPNT (500 ng/ml) for 24 hours. Scale bar, 100 μm. ( D , E ) Quantitative analysis of branch points and tube lengths. PBS and bFGF were used as a negative and positive control respectively. ( F ) Representative images showing that recombinant mouse NPNT (200 ng/ml) induced vessel outgrowth from metatarsals dissected from E17.5 embryos. Scale bar, 250 μm. ( G ) Quantitative analysis of vessel sprouting. PBS and VEGF (50 ng/ml) were used as a negative and positive control respectively. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant mouse NPNT (R&D Systems), human bFGF (PeproTech, Inc.), human VEGF (PeproTech, Inc.) and ERK1/2 inhibitor U0126 (Promega) were purchased for the assays.

Techniques: Recombinant, Migration, Positive Control

Journal: Scientific Reports

Article Title: NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

doi: 10.1038/srep36210

Figure Lengend Snippet: ( A ) Western blot images showing the treatment of SVEC cells by mouse recombinant NPNT resulted in the phosphorylation of ERK1/2 and p-38, but not Akt. β-Actin was used as a loading control. ( B – D ) Quantification of signal intensities of p-ERK1/2, p-p38 and p-Akt by ImageJ. Induction ratios at each timepoint were compared to 0 minute, with p-ERK1/2 normalized to ERK1/2, p-p38 normalized to p-38 and p-Akt normalized to Akt. Western blot images are presented as cropped format. Full length blots are presented in . *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant mouse NPNT (R&D Systems), human bFGF (PeproTech, Inc.), human VEGF (PeproTech, Inc.) and ERK1/2 inhibitor U0126 (Promega) were purchased for the assays.

Techniques: Western Blot, Recombinant, Phospho-proteomics, Control

Journal: Scientific Reports

Article Title: NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

doi: 10.1038/srep36210

Figure Lengend Snippet: ( A ) Representative microscopic images of scratch wound healing assays showing NPNT-induced endothelial cell migration was blocked in the presence of U0126 (5 μM). Scale bar, 100 μm. Quantitative analyses showing that ( B ) NPNT-induced endothelial cell migration and ( C ) tube-like structure formation were significantly inhibited by U0126. PBS and bFGF were used as a negative and positive control, respectively. ( D ) Representative images showing that NPNT-induced angiogenesis was inhibited in the presence of U0126 (5 μM). Scale bar, 250 μm. ( E ) Quantitative analysis of vessel sprouting. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant mouse NPNT (R&D Systems), human bFGF (PeproTech, Inc.), human VEGF (PeproTech, Inc.) and ERK1/2 inhibitor U0126 (Promega) were purchased for the assays.

Techniques: Migration, Positive Control

Journal: medRxiv

Article Title: Development of mass spectrometry-based targeted assay for direct detection of novel SARS-CoV-2 coronavirus from clinical specimens

doi: 10.1101/2020.08.05.20168948

Figure Lengend Snippet: Overview of the annotated genome (A) and domain organization of key structural components: nucleocapsid and spike proteins (B) of SARS-CoV-2 along with sequence coverage obtained by peptides identified by mass spectrometry in this study. Overview of proteins and peptides from SARS-CoV-2 identified in discovery studies by bottom-up mass spectrometry (C). Red boxes represent peptides that were detected while unfilled boxes represent peptides that were not detected in the indicated samples.

Article Snippet: Recombinant SARS-CoV-2 nucleocapsid protein (97-077) was purchased from ProSci (Fort Collins, CO).

Techniques: Sequencing, Mass Spectrometry

Journal: medRxiv

Article Title: Development of mass spectrometry-based targeted assay for direct detection of novel SARS-CoV-2 coronavirus from clinical specimens

doi: 10.1101/2020.08.05.20168948

Figure Lengend Snippet: Sequence alignment of SARS-CoV-2 nucleocapsid protein against related coronaviruses: SARS-CoV, MERS and common human coronaviruses (L229E, NL63, HKU1 and OC43). Sequence in red indicates the regions that were identified by mass spectrometry while the underlined sequences were selected for targeted PRM analysis.

Article Snippet: Recombinant SARS-CoV-2 nucleocapsid protein (97-077) was purchased from ProSci (Fort Collins, CO).

Techniques: Sequencing, Mass Spectrometry

Journal: medRxiv

Article Title: Development of mass spectrometry-based targeted assay for direct detection of novel SARS-CoV-2 coronavirus from clinical specimens

doi: 10.1101/2020.08.05.20168948

Figure Lengend Snippet: Experimental annotated MS/MS spectra of peptides identified from the nucleocapsid protein of SARS-CoV-2 positive nasopharyngeal swab specimens are shown. GQGVPINTNSSPDDQIGYYR (A), NPANNAAIVLQLPQGTTLPK (B), AYNVTQAFGR (C) and QQTVTLLPAADLDDFSK (D). Sequence alignment of the four most abundant peptides that were chosen for development of PRM assays (E) - GQGVPINTNSSPDDQIGYYR (69-88), NPANNAAIVLQLPQGTTLPK (150-169), AYNVTQAFGR (267-276) and QQTVTLLPAADLDDFSK (389-405) derived from SARS-CoV-2 nucleocapsid protein across related coronaviruses (SARS-CoV, MERS and common human coronaviruses: L229E, NL63, HKU1 and OC43). Amino acid mismatches are indicated in red while tryptic cleavage sites (R/K) are underlined.

Article Snippet: Recombinant SARS-CoV-2 nucleocapsid protein (97-077) was purchased from ProSci (Fort Collins, CO).

Techniques: Tandem Mass Spectroscopy, Sequencing, Derivative Assay

Journal: medRxiv

Article Title: Development of mass spectrometry-based targeted assay for direct detection of novel SARS-CoV-2 coronavirus from clinical specimens

doi: 10.1101/2020.08.05.20168948

Figure Lengend Snippet: A schematic of FAIMS-PRM targeted assay for the detection of diagnosis of SARS-CoV-2. Heat-inactivated nasopharyngeal swab samples were immunopurified with anti-nucleocapsid antibody coated on MSIA D.A.R.T.s™ tips using Versette automated liquid handler, in-solution trypsin digested. Targeted LC-MS/MS analysis was carried out on Exploris 480 mass spectrometer interfaced with rapid chromatography system and a FAIMS Pro interface.

Article Snippet: Recombinant SARS-CoV-2 nucleocapsid protein (97-077) was purchased from ProSci (Fort Collins, CO).

Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Chromatography

Journal: Molecular and Cellular Biology

Article Title: Sortilin Facilitates Signaling of Ciliary Neurotrophic Factor and Related Helical Type 1 Cytokines Targeting the gp130/Leukemia Inhibitory Factor Receptor β Heterodimer ▿

doi: 10.1128/MCB.00274-10

Figure Lengend Snippet: Sortilin binds and enhances the signaling of CLC/CLF-1 and neuropoietin. (A) SPR analysis of CLC/CLF-1 (left) and neuropoietin (NP) (right) binding to immobilized s-sortilin. The responses obtained in both the absence and presence of NT-mediated inhibition are shown, and the estimated Kd values are indicated. (B) Binding of CLC/CLF-1 in the absence or presence of a 200-fold excess of the 13-residue peptide constituting the C terminus of CNTF. (C) STAT3 phosphorylation induced by CLC/CLF-1 (left) and neuropoietin (right). BA/F3 transfectants expressing the indicated combinations of receptors were stimulated for 15 min at 37°C with CLC/CLF-1 (40 nM) plus sCNTFRα (16 nM) or neuropoietin (1 nM) prior to Western blotting of lysed cells.

Article Snippet: Recombinant human CLC/CLF-1, CT-1, OSM, sCNTFRα, soluble LIFRβ (sLIFRβ), recombinant mouse neuropoietin, and goat anti-CNTFRα were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Inhibition, Residue, Phospho-proteomics, Expressing, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Sortilin Facilitates Signaling of Ciliary Neurotrophic Factor and Related Helical Type 1 Cytokines Targeting the gp130/Leukemia Inhibitory Factor Receptor β Heterodimer ▿

doi: 10.1128/MCB.00274-10

Figure Lengend Snippet: Affinity of helical type 1 cytokine binding to immobilized s-sortilin determined by Biacore analysis

Article Snippet: Recombinant human CLC/CLF-1, CT-1, OSM, sCNTFRα, soluble LIFRβ (sLIFRβ), recombinant mouse neuropoietin, and goat anti-CNTFRα were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Nephronectin promotes breast cancer brain metastatic colonization via its integrin-binding domains

doi: 10.1038/s41598-020-69242-1

Figure Lengend Snippet: The integrin-binding sites of NPNT are important for adhesion. ( a ) bEND.3 cells were seeded onto rmNPNT- or pluronic coated surfaces for 0, 1, 3, 5 or 24 h. Cell lysates were harvested and analysed by Western blotting for tyrosine phosphorylation. ( b ) bEND.3 cells were seeded onto rmNPNT- or Pluronic coated surfaces for 3 h. Harvested cell lysates were analysed for specific tyrosine phosphorylated proteins using the Proteome Profiler antibody array (human Phospho-kinase Array). ( c ) Adhesion of 66cl4-EV cells to rmNPNT was analysed by real-time cell analysis using the xCELLigence system. The experiments were performed at least twice with similar results and with two technical replicates per run. Impedance created by adhering cells gave the arbitrary “cell index” value that is proportional to the amount of adhered cells. Adhesion was recorded every 15 min for 12 h. Wells were coated with either 3% BSA as a negative control or 10 μg purified wild-type rmNPNT (Wild-type) or rmNPNT mutated in the integrin-binding sites (Mutant). Error bars show the pooled standard deviation. ( d ) Wells were coated with 10 μg purified wild-type rmNPNT. Adhesion of 66cl4-EV cells in the presence of RGD-blocking peptide or scrambled negative control peptide. p = 0.019. ( e ) 66cl4 cells were seeded onto a confluent monolayer of MBECs. After three hours, non-adherent cells were washed off and adherent cells were counted. Fold differences between the cells is shown in the graph (N = 2, n = 3). p = 0.013. ( f ) 66cl4 cells were seeded onto a confluent monolayer of bEND.3 cells. After three hours, non-adherent cells were washed off and adherent cells were counted. Fold differences between the cells is shown in the graph (N = 3, n = 3). p = NS. ( g ) 66cl4 cells were seeded onto a confluent monolayer of MBECs. Transmigration of 66cl4 cells was followed by imaging every 5 min for 24 h. Every cell that transmigrated through the monolayer was recorded and fold differences between the cells is presented in the graph (N = 3, n = 3), p = 0.044.

Article Snippet: E-Plates were coated with either purchased rmNPNT (Cat: 4,298-NP-050, RD Systems, Minneapolis, MN) or purified wild-type rmNPNT (wt) or mutated rmNPNT (mutant).

Techniques: Binding Assay, Western Blot, Phospho-proteomics, Ab Array, Cell Analysis, Negative Control, Purification, Mutagenesis, Standard Deviation, Blocking Assay, Transmigration Assay, Imaging

Journal: Biochemical pharmacology

Article Title: Biochemical pathways of 8-aminoguanine production in Sprague-Dawley and Dahl salt-sensitive rats.

doi: 10.1016/j.bcp.2022.115076

Figure Lengend Snippet: Fig. 17. Evidence supporting the role of purine nucleoside phosphorylase (PNPase) in the biosynthetic pathway for production of 8-aminoguanine from 8-nitrogua nosine. Red indicates role of PNPase is an open question; EVIDENCE represents findings that address the role of PNPase; green indicates role of PNPase is confirmed. Also shown are sources of 8-nitroguanosine and the possible contribution of guanine to 8-nitroguanine formation. Not shown are possible contributions of 8-nitro-2ˊ- deoxyguanosine from DNA (or directly from 2ˊ-deoxyguanosine) to the formation of 8-aminoguanine. 8-NO2-GS, 8-nitroguanosine; 8-NO2-GN, 8-nitroguanine; 8- NH2-GS, 8-aminoguanosine; 8-NH2-GN, 8-aminoguanine.

Article Snippet: 8-Aminoguanine, 8-aminoguanosine, 8-nitroguanine and 8-nitroguanosine were from Toronto Research Chemicals (Toronto, Ontario, Canada); guanine, xanthine and guanosine were from Sigma-Aldrich (St. Louis, MO); 13C2,15N-8-aminoguanosine and 13C2,15N-8-aminoguanine were from Medical Isotopes (Pelham, NH); recombinant human purine nucleoside phosphorylase (PNPase) was from R&D Systems (Minneapolis, MN); and recombinant human guanase was obtained from Novus Biologicals (Centennial, CO).

Techniques: