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The proteins p38 and phospho-p38 were detected using rabbit anti-human p38 (Cell Signaling, USA) and mouse anti-human phospho-p38 (Cell Signaling, USA) with the secondary antibodies goat anti-mouse IRDye800 (Molecular Probes, USA) and goat anti-rabbit AlexaFluor680 (Molecular Probes, USA). Blots were scanned using GUCY1B3 the Odyssey? Infrared Imaging System (Li-Cor, USA) at 700 and 800?nm and analyzed using Odyssey? software v1.2 (Li-Cor, USA). Cytokine concentrations were determined in moDCs or co-culture supernatants using commercial ELISAs specific for IFN-�� IL-8 (both BioSource, Solingen, Germany), and IL-12p70 (eBioscience, San Diego, CA, USA) according to manufacturer��s instructions. After differentiation, moDCs were recultured and incubated with FITC-labeled latex beads (Polysciences, USA) in ��-slide VI (Ibidi GmbH, Germany). Cells were fixed with Selleck GSK2879552 4% PFA, permeabilized with 0.25% Triton X-100, and dyed with mouse anti-human TLR2 (Genentech, USA) and TOTO-3-iodid (Invitrogen, Germany). Slides were analyzed using the laser scanning microscope TCS SP and the software TCS NT (Leica, Germany). Stimulated moDCs were harvested and stained with fluorochrome-conjugated mAbs for CD40 (PE-labeled, BeckmanCoulter, USA), CD80 (PE-labeled, BeckmanCoulter, USA), CD86 (PE-labeled, BD PharMingen, USA), or MHC class II (PE-labeled, BeckmanCoulter, USA) and were fixed with 1.5% paraformaldehyde. Isotype controls IgG1 (PE-labeled, BeckmanCoulter, USA) and IgG2a (PE-labeled, BeckmanCoulter, USA) were used in parallel. Data analysis was performed with the WinMDI 2.9 software developed by Joseph Trotter (Scripps Institute, La Jolla, CA, USA). Statistical analysis (paired t-test, one-tailed P-value) was performed with GraphPad Prism5. Data presented in the Figs?1A, B and 5A, C are presented as group data from KD025 supplier the indicated number of subjects. Significant differences compared to control cells are indicated by *(P?