Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-beta1a inhibits the secretion of Th17-polarizing cytokines in human dendritic cells via TLR7 up-regulation.

doi: 10.4049/jimmunol.0802226

Figure Lengend Snippet: FIGURE 2. IFN-1a treatment up-regulates TLR7 and its signaling pathway, while it down-regulates the expression of IL-1R and IL-1/. A, Three 106 DCs per condition derived from 10 RR MS patients were pretreated with IFN-1a for 24 h and then matured with LPS for 2 h before RNA extraction. TLR7, MyD88, IRAK4, TRAF6, IL-1R1, IL-1, and IL-1 gene expression was measured by RT- PCR. The results are presented as relative gene expression normalized for the 18S RNA expression. B, Three 106 DCs per condition from 16 RR MS patients were pretreated with IFN-1a for 24 h and then matured with LPS for 48 h. DCs were stained with PE-Cy-CD11c, allophycocyanin-CD1a mAb, and anti-IL-1R1 Ab, followed by FITC-conjugated secondary Ab, anti-TLR7 primary Ab, and FITC-conjugated secondary Ab. FITC-conjugated IgG isotype Ab was used as a control. The percentage of positive cells was determined in the CD1a-gated population. C, Three 106 DCs per condition derived from three RR MS patients were pretreated by IFN-1a for 24 h and then matured with LPS for 5 h before protein extraction. The expression of TLR7, MyD88, IRAK4, TRAF6, IL-1R1, and -actin were measured by Western blotting. The results present one of three similar experiments.

Article Snippet: Nonspecific binding was blocked for 1 h in the blocking buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 2% fish gelatin, and 1% OVA in double-distilled water) and incubated overnight with rabbit anti-human Ab for TLR7 (Abcam), MyD88, TRAF6 (Santa Cruz Biotechnology), and IRAK4 (ProSci) and with mouse anti-human Ab for IL1R1 and -actin (Santa Cruz Biotechnology).

Techniques: Expressing, Derivative Assay, RNA Extraction, Gene Expression, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Staining, Control, Protein Extraction, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-beta1a inhibits the secretion of Th17-polarizing cytokines in human dendritic cells via TLR7 up-regulation.

doi: 10.4049/jimmunol.0802226

Figure Lengend Snippet: FIGURE 4. IFN-1a modifies MyD88/IRAK4/ TRAF6 signaling and cytokine production in DCs through its induction of TLR7 expression. Three 106/well DCs generated from six RR MS patients were plated in antibiotic-free medium. After a 24-h incubation at 37oC, the DCs were transfected with TLR7 siRNA or control siRNA. The siRNA-treated cells were harvested and cultured in serum-free me- dium in the absence or presence of IFN-1 for 24 h, followed by LPS maturation for 5 h before the pro- tein extraction, and for 48 h before the SN collec- tion. A, The expression of TLR7, MyD88, IRAK4, TRAF6, IL-1R1, and -actin was measured by Western blotting. The results present one of three similar experiments. B, The production of IL-1, TGF-1, IL-23/p19, and IL-27 was measured by ELISA. The results present six experiments per- formed in triplicate. Statistical analysis was per- formed using a repeated measures ANOVA. , p 0.05; , p 0.01; and , p 0.001.

Article Snippet: Nonspecific binding was blocked for 1 h in the blocking buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 2% fish gelatin, and 1% OVA in double-distilled water) and incubated overnight with rabbit anti-human Ab for TLR7 (Abcam), MyD88, TRAF6 (Santa Cruz Biotechnology), and IRAK4 (ProSci) and with mouse anti-human Ab for IL1R1 and -actin (Santa Cruz Biotechnology).

Techniques: Expressing, Generated, Incubation, Transfection, Control, Cell Culture, Extraction, Western Blot, Enzyme-linked Immunosorbent Assay