Journal: Scientific Reports

Article Title: Identification of potential autoantigens in anti-CCP-positive and anti-CCP-negative rheumatoid arthritis using citrulline-specific protein arrays

doi: 10.1038/s41598-021-96675-z

Figure Lengend Snippet: Immunome slide design and protein categories on the protein microarray. ( A ) The Immunome protein microarray consists of four identical subarrays, each containing 1631 different proteins in addition to 11 control proteins (BCCP, BSA, Cy3BSA, IgA, IgG, IgM, and four different control probes for the BCCP tag acting as negative controls). The control proteins are located between the subarrays. ( B ) The proteins spotted on the microarray represent different protein groups, such as cancer-associated antigens, transcription factors, kinases, and signaling proteins. The number of proteins in each category is shown. BCCP biotin carboxyl carrier protein, BSA bovine serum albumin.

Article Snippet: Plasma from anti-CCP-positive or anti-CCP-negative RA patients was incubated with Immunome protein microarray slides containing native proteins or proteins citrullinated by PAD2 or PAD4.

Techniques: Microarray

Journal: Microbiology Spectrum

Article Title: Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes

doi: 10.1128/spectrum.02452-21

Figure Lengend Snippet: Immune recognition of various EV-D68 densities and characterization of membrane-associated virus. (A) The y axis represents average dilutions of anti-EV-D68 mouse serum required to neutralize virus, divided by the average TCID 50 for respective viral densities (ANOVA post hoc Student'’s t test, P = 0.42, P = 0.68, P = 0.70). (B) Three viral density isolates (1.11, 1.20, and 1.24 g/cm 3 ) were treated with 0.01 mg/mL 15C5-Chmra antibody for 1 h, then mix was put onto TCID 50 plates to assess the viral titer of each isolate. Gray highlight represents detection limit. Asterisks (*) indicate statistical significance (1.11 g/cm 3 , P = 0.0003; 1.20 g/cm 3 , P < 0.0001; 1.24 g/cm 3 , P = 0.0064), all versus respective control, determined by Dunnett’s Method. (C) 15C5-Chmra antibody bound to magnetic beads was added to membrane-associated and naked virus. After 1 h, a magnet was used to remove antibody and the supernatant was added to a TCID 50 plate to assess viral titer (15C5-Chmra versus control: *, P < 0.0005 for both membrane-associated and naked virus; Dunnett’s Method). (D) ICAM-5 or N -acetylneuraminic acid (sialic acid) were attached to magnetic beads and the antibody/bead complex was incubated with membrane-associated or naked virus samples for 1 h. Beads were rinsed twice in excess PBS and viral titer was assessed to determine how much virus was immunoprecipitated from the supernatant (control versus ICAM5 and control versus sialic acid for membrane-associated and naked virus; *, P = 0.0001 determined by Dunnett’s Method). (E) Exosome antibody array on 1.11 g/cm 3 fraction, examining cytosolic proteins (FLOT1, ALIX, TSG101), transmembrane proteins (CD63, CD81, ANXA5), and cis -golgi matrix protein as markers for cellular contamination (GM130). Example blot is shown on the right and chart represents average intensity across three biological replicates. Positive control indicates detection reagents are working correctly, and do not represent an exosome-specific control. Error bars represent standard deviation. Statistics: comparison with control (blank) using Dunnett’s Method ( P = 0.999 for GM130; *, P = 0.027 for FLOT1; P = 0.218 for ICAM; *, P = 0.005 for ALIX; P = 0.086 for CD81; *, P < 0.0001 for CD63; P = 0.305 for EpCAM; *, P < 0.0001 for ANXA5; *, P = 0.0008 for TSG101). Asterisks indicate statistical significance. (F) Anti-CD81 or anti-CD63 antibodies were attached to magnetic beads and incubated with membrane-associated virus. Supernatant was discarded, and beads were rinsed and treated with 0.01% NP-40 (to dissolve exosomes and release virus from bead) before TCID 50 measurement. CD81 versus control: *, P = 0.0178; CD63 versus control: *, P = 0.0180 as determined by Dunnett’s Method. Gray highlight represents detection limit. (G) RD or SH-SY5Y cells in TCID 50 plate were infected with MO47 with or without exosomes in the medium. The “A549 exosomes added” bar represents exosome-depleted media to which purified A549 exosomes were added. Gray panel represents TCID 50 plates containing SH-SY5Y cells. Each condition represents 3 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05. Green panel represents TCID 50 plates containing RD cells. Each condition represents 4 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05.

Article Snippet: We followed the coupling protocol from the Dynabeads Antibody Coupling Kit (Thermo Fisher, cat no. 14311D) to covalently attach magnetic beads to the following antibodies: anti-CD81 (1D6) monoclonal antibody (Novus Biologicals NB100-65805), anti-CD63 (H5C6) monoclonal antibody (Novus Biologicals NBP2-42225), 15C5-chimeric monoclonal antibody (generous gift from Michael Pauly at ZabBio), and anti-HSV negative control (generous gift from Michael Pauly at ZabBio).

Techniques: Magnetic Beads, Incubation, Immunoprecipitation, Ab Array, Positive Control, Standard Deviation, Infection, Purification

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