Transam flexi nf κb family kit

Indoleamine 2,3-dioxygenase 1 protein expression was investigated in pDCs by immunoblot with a rabbit polyclonal anti-mouse IDO1 antibody raised in our laboratory (20 (link)). Anti-β-tubulin antibody (Sigma-Aldrich) was used as a normalizer. Mouse IL-6, IL-17A, IFN-γ, IL-10, and TGF-β1 were measured in culture supernatants by ELISA with specific kits (eBioscience, Inc., San Diego, CA, USA; Promega Italia, S.r.l., Milano, Italy). An ELISA–based TransAM Flexi NF-κB Family Kit (Active Motif, Rixensart, Belgium) was used to monitor activity of NF-κB family members, as described (6 (link), 7 (link), 10 (link)). Cytofluorimetric assays were conducted using the specific fluorochrome-labeled antibodies for murine CD19, CD5, CD1d, CD4, Foxp3, and RORγt (Biolegend, BD Pharmingen, eBiocience Inc., San Diego, CA, USA; Miltenyi Biotech, Germany), as described (6 (link)). For intracellular staining of Foxp3 and RORγt, cells were previously permeabilized by the Fix/Perm Buffer set (Biolegend, San Diego, CA, USA).

Mouse IL-6, IL-10, IL-27, TNF-α and IFN-α were measured in culture supernatants by ELISA with specific kits (eBioscience, Inc., San Diego, CA, USA; Promega Italia, S.r.l., Milano, Italy; and pbl Assay Science, Piscataway, NJ, USA). An ELISA-based TransAM Flexi NF-κB Family Kit (Active Motif, Rixensart, Belgium) was used to monitor activity of NF-κB family members, as described 9 (link),20 (link),25 (link). Cytofluorometric assays were conducted by using FITC-labelled anti-latency-associated peptide (LAP) TGF-β1 (clone TWA-2F8; BioLegend, San Diego, CA, USA) and PE-labelled anti-B220 antibodies (BD Pharmingen, San Diego, CA, USA) as described 28 (link).