mouse plasma cytokine array mouse plasma cytokine antibody array analyses  (R&D Systems)

Journal: Journal of Biological Chemistry

Article Title: Interleukin-35 Inhibits Endothelial Cell Activation by Suppressing MAPK-AP-1 Pathway

doi: 10.1074/jbc.m115.663286

Figure Lengend Snippet: FIGURE 3. IL-35 suppresses cytokine and chemokine expression in the plasma of wild type mice challenged with LPS. A, in the left panel, mouse cytokine/chemokine arrays were performed to detect the expression changes of 40 inflammatory cytokines/chemokines in the plasma from mice treated with LPS, LPS plus rIL-35, and WT control mice. Plasma samples in each group were pooled from three mice. In the right panel, the arrangement of the mouse cytokine/chemokine array was shown. Cytokines and chemokines that were induced by LPS are in bold. rIL-35-inhibited cytokines and chemokines are labeled with asterisks. B and C, quantification of cytokine and chemokine expressions. The variations of the manufacturer’s designated positive control spots between each array were used to determine the confidence interval of nonspecific variations between samples. *, p 0.05.

Article Snippet: Mouse Plasma Cytokine Array—Mouse plasma cytokine antibody array analyses were carried out according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Clinical Proteomics, Control, Labeling, Positive Control

Journal: Journal of Biological Chemistry

Article Title: Interleukin-35 Inhibits Endothelial Cell Activation by Suppressing MAPK-AP-1 Pathway

doi: 10.1074/jbc.m115.663286

Figure Lengend Snippet: FIGURE 6. IL-35 inhibits LPS-induced activation of MAPK-AP1 pathway, which mediates LPS-induced VCAM-1 up-regulation in human aortic endo- thelial cells. A, the expressions of proteins involved in the MAPK pathway were analyzed with Western blots in HAECs stimulated by LPS and LPS plus IL-35 treatments for 5, 15, and 30 min. The relative mean pixel density ratios of phosphorylated (p) kinase over total kinase proteins were shown. B, HAECs were treated with LPS for 1.5 h with or without IL-35 pretreatment for 18 h, and EMSA was used to detect the binding of transcription factor AP-1 in the nuclear extracts. C, EMSA was used to determine the specificity of AP-1 binding to the AP-1 consensus oligonucleotides using consensus competitor oligonucleotides, mutant competitor oligonucleotides, and supershift antibodies for AP-1 subunits c-Fos and c-Jun. D, AP-1-binding sites were found in the promoters of VCAM-1 gene, proinflammatory cytokine genes, and chemokine genes that were suppressed by IL-35. N.D., not determined; N.F., not found; PMID, PubMed IDs of the studies. E, protein expressions of phosphorylated (p)-IB, total IB, and -actin were analyzed using Western blots. The relative mean pixel density ratios of phosphorylated over total proteins were shown. F, EMSAs were used to detect the binding of transcription factor NF-B in the nuclear extracts in HAECs. G, EMSA was used to determine the specificity of NF-B binding to the NF-B consensus oligonucleotide using competitor oligonucleotides, mutant oligonucleotides, and supershift antibodies for NF-B subunits p50 and p65. The result shown is representative of 3 independent experiments.

Article Snippet: Mouse Plasma Cytokine Array—Mouse plasma cytokine antibody array analyses were carried out according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Techniques: Activation Assay, Western Blot, Binding Assay, Mutagenesis