Rabbit anti human irf5 antibody

For analysis of TBK1/IKKε phosphorylation, DCs or macrophages were stimulated as indicated in 48-well plates (Greiner Bio-One) for 30 min and fixed using Lyse/Fix buffer (BD Biosciences) for 10 min at room temperature. For analysis of IRF5, unstimulated DCs were also lysed and transferred in a 96-well plate following the same protocol as TBK1 phosphorylation. Cells were harvested by gentle scraping, transferred to a 96-well round-bottom plate (Greiner Bio-One), washed in PBS, and permeabilized using Perm III buffer (BD Biosciences) for at least 30 min at −20°C. Cells were then washed in PBS containing 0.5% BSA and 0.1% sodium azide and stained for 1 h at RT with a rabbit-anti-human-IRF5 antibody (1:200) (Cell Signaling) or a rabbit-anti-human-pTBK1 antibody (1:50) (Ser172; Cell Signaling), which also reacts to pIKKε, followed by a 30 min staining at room temperature with Alexafluor488-labeled goat-anti-rabbit-IgG antibody (1:400) (Molecular Probes). Fluorescence was determined by flow cytometry (Canto II, BD Biosciences).

For analysis of IRF5 translocation, DCs or macrophages were stimulated as indicated in Maxisorp plates. After 2 h stimulation, cells were washed with PBS, fixed with 3.7% formaldehyde (Sigma-Aldrich) for 15 min at room temperature, washed in PBS and stored in PBS containing 0.5% bovine serum albumin (BSA; PAA) and 0.1% sodium azide (Merck) at 4°C. Cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 5 min at room temperature and blocked for 30 min in PBS containing 0.5% BSA and 0.1% sodium azide. Cells were then stained with a rabbit-anti-human-IRF5 antibody (1:400) (Cell Signaling) or rabbit-anti-human NF-kB p65 antibody (1:100) (Cell Signaling) for 45 min at room temperature, washed with PBS and stained with a Cy3-labeled goat-anti-rabbit-IgG antibody (1:50) (Jackson ImmunoResearch). Cells were again washed with PBS and nuclei were stained using 1 μg/mL Hoechst (Immunochemistry Technologies) for 1 min at room temperature. Cells were imaged using a DM IRB inverted fluorescence microscope (Leica), combined with a DFC 300FX digital color camera (Leica).