c di amp standard  (MedChemExpress)

Journal: Veterinary Research

Article Title: The c-di-AMP binding protein NadD from Mesomycoplasma ovipneumoniae functions as a phosphodiesterase that inhibits host inflammatory responses

doi: 10.1186/s13567-025-01707-5

Figure Lengend Snippet: c-di-AMP production during M. ovipneumoniae infection of sheep alveolar macrophages . A Growth curve of M. ovipneumoniae . B–D ESI‒MS analysis of relevant HPLC fractions from M. ovipneumoniae cellular extracts and culture supernatants; B c-di-AMP standard; C M. ovipneumoniae intracellular extracts; D M. ovipneumoniae culture supernatants. E–G Flow cytometry was used to detect the specific surface antigen CD14 on primary sheep alveolar macrophages, with OAR-L1 sheep lung fibroblasts used as a control. E Flow cytometric collection diagram for OAR-L1 sheep lung fibroblasts. F Flow cytometric collection diagram for primary sheep alveolar macrophages. G Flow cytometric analysis diagram of OAR-L1 sheep lung fibroblasts and primary alveolar macrophages. M1 indicates the fluorescently labelled negative interval, whereas M2 represents the fluorescently labelled positive interval. H Fluorescence observation of M. ovipneumoniae -infected sheep alveolar macrophages. Blue: DAPI; green: DiO-labelled M. ovipneumoniae ; red: phalloidin-labelled cytoskeleton. I Quantitative analysis of intracellular c-di-AMP during M. ovipneumoniae infection of sheep alveolar macrophages. The means ± SDs of 3 independent experiments were compared by one-way ANOVA.

Article Snippet: The c-di-AMP standard was purchased from MCE, the 5'-pApA standard was purchased from BioLog, and the AMP standard was purchased from Sigma.

Techniques: Infection, Flow Cytometry, Control, Fluorescence

Journal: Veterinary Research

Article Title: The c-di-AMP binding protein NadD from Mesomycoplasma ovipneumoniae functions as a phosphodiesterase that inhibits host inflammatory responses

doi: 10.1186/s13567-025-01707-5

Figure Lengend Snippet: Transcriptomic analyses reveal that c-di-AMP activates immune responses in primary sheep alveolar macrophages . A The FPKM violin plot displays gene expression across each sample. B PCA of the samples is presented. C A volcano plot highlights the differentially expressed genes in the M. ovipneumoniae group versus the control group. D Volcano plot showing the differentially expressed genes in the c-di-AMP group versus the control group. E GO enrichment analysis identified genes whose expression was altered in the M. ovipneumoniae and c-di-AMP groups compared with the control group. F KEGG enrichment analysis of the genes whose expression changed in the M. ovipneumoniae and c-di-AMP groups relative to that in the control group. G RT‒qPCR verification of differentially upregulated genes in the transcriptome. H RT‒qPCR verification of differentially downregulated genes in the transcriptome. The means ± SDs of 3 independent experiments were compared by one-way ANOVA. Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: The c-di-AMP standard was purchased from MCE, the 5'-pApA standard was purchased from BioLog, and the AMP standard was purchased from Sigma.

Techniques: Gene Expression, Control, Expressing

Journal: Veterinary Research

Article Title: The c-di-AMP binding protein NadD from Mesomycoplasma ovipneumoniae functions as a phosphodiesterase that inhibits host inflammatory responses

doi: 10.1186/s13567-025-01707-5

Figure Lengend Snippet: Specific binding of c-di-AMP to the NadD protein of M. ovipneumoniae . A The c-di-AMP-binding proteins were identified by silver staining. Control: agarose coupled with ethanolamine; c-di-AMP: agarose coupled with c-di-AMP. Silver staining was performed in 2 independent experiments. B A Venn diagram depicting the identification of the spectrum of c-di-AMP-binding proteins. C The identification of the purified NadD protein by SDS‒PAGE analysis. Lane 1: the empty vector before induction; lane 2: the empty vector after 16 h of induction; lane 3: the pET-28b-NadD vector prior to induction; lane 4: the pET-28b-NadD vector after 16 h of induction; lane 5: the supernatant protein following the 16-h induction of the pET-28b-NadD vector; lane 6: the running-off liquid; lane 7: the washing solution; lane 8: the elution solution. D Western blot analysis confirmed the presence of the purified NadD protein. Lane 1: the empty vector before induction; lane 2: the empty vector after 16 h of induction; lane 3: the pET-28b-NadD vector before induction; lane 4: the pET-28b-NadD vector after 16 h of induction; lane 5: the supernatant protein after the 16-h induction of the pET-28b-NadD vector; lane 6: the elution mixture. E Silver staining results demonstrating the binding of the recombinant NadD protein to c-di-AMP. M: marker; lane 1: NadD recombinant protein; lane 2: running-off of the control group; lane 3: running-off of the c-di-AMP group; lane 4: washing solution II of the control group; lane 5: washing solution II of the c-di-AMP group; lane 6: washing solution III of the control group; lane 7: washing solution III of the c-di-AMP group; lane 8: eluate of the control group; lane 9: eluate of the c-di-AMP group. F The concentration gradient binding curve between NadD and c-di-AMP is shown, as detected by SPR.

Article Snippet: The c-di-AMP standard was purchased from MCE, the 5'-pApA standard was purchased from BioLog, and the AMP standard was purchased from Sigma.

Techniques: Binding Assay, Silver Staining, Control, Purification, Plasmid Preparation, Western Blot, Recombinant, Marker, Concentration Assay

Journal: Veterinary Research

Article Title: The c-di-AMP binding protein NadD from Mesomycoplasma ovipneumoniae functions as a phosphodiesterase that inhibits host inflammatory responses

doi: 10.1186/s13567-025-01707-5

Figure Lengend Snippet: Biochemical characterization of NadD as a phosphodiesterase . A Analysis of the conserved domains of NadD. B–H LC‒MS analysis of the hydrolysis of c-di-AMP and pApA by NadD. B – D depict the standard, while E – F show the enzymatic reaction products of NadD with c-di-AMP, and G-H illustrate the enzymatic reaction products of NadD with pApA. I HPLC analysis of c-di-AMP hydrolysis by NadD at various pH values. J HPLC analysis of NadD-mediated hydrolysis of c-di-AMP in the presence of different metal ions. Three independent experiments were repeated by HPLC.

Article Snippet: The c-di-AMP standard was purchased from MCE, the 5'-pApA standard was purchased from BioLog, and the AMP standard was purchased from Sigma.

Techniques:

Journal: Veterinary Research

Article Title: The c-di-AMP binding protein NadD from Mesomycoplasma ovipneumoniae functions as a phosphodiesterase that inhibits host inflammatory responses

doi: 10.1186/s13567-025-01707-5

Figure Lengend Snippet: NadD suppresses inflammatory responses in host cells infected with M. ovipneumoniae . A Western blot analysis and quantification of NadD and DacM proteins during M. ovipneumoniae infection of sheep alveolar macrophages. B Fluorescence observations (left) and western blot results (right) for the cells transfected with the pEGFP-C1 and pEGFP-C1-NadD plasmids. C RT‒qPCR analysis of the effect of the NadD protein on the mRNA expression of inflammatory factors during M. ovipneumoniae infection of sheep alveolar macrophages. NC + M. ovipneumoniae , NadD + M. ovipneumoniae , NC + c-di-AMP, and NadD + c-di-AMP represent the conditions where sheep alveolar macrophages were first transfected with an empty plasmid (NC) or the NadD plasmid, followed by infection with M. ovipneumoniae or treatment with c-di-AMP. The means ± SDs of 3 independent experiments were compared by one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: The c-di-AMP standard was purchased from MCE, the 5'-pApA standard was purchased from BioLog, and the AMP standard was purchased from Sigma.

Techniques: Infection, Western Blot, Fluorescence, Transfection, Expressing, Plasmid Preparation

Journal: Veterinary Research

Article Title: The c-di-AMP binding protein NadD from Mesomycoplasma ovipneumoniae functions as a phosphodiesterase that inhibits host inflammatory responses

doi: 10.1186/s13567-025-01707-5

Figure Lengend Snippet: A schematic diagram of the c-di-AMP molecule and the phosphodiesterase NadD protein from M. ovipneumoniae . The NadD protein specifically binds to c-di-AMP and exhibits phosphodiesterase activity, which can inhibit the transcription of various inflammatory factors induced by M. ovipneumoniae .

Article Snippet: The c-di-AMP standard was purchased from MCE, the 5'-pApA standard was purchased from BioLog, and the AMP standard was purchased from Sigma.

Techniques: Activity Assay