Nuclear factor kappa B (NF‐κB) translocation

Cells were grown on 9 cm² dishes, treated for 24 h and infected as described above. Extraction of the nuclear and cytoplasmic fraction was performed with the NE‐PER Nuclear and Cytoplasmic Extraction Reagents following the manufacturer’s protocol (Thermo Scientific). Starting material was a cell pellet of approximately 20 µl per sample. The protein concentration in the final extracts was determined using the BCA assay (Pierce, Thermo Scientific) and samples were stored at –80°C until analysis. An equal amount of protein in 15 µl buffer was mixed with an equal volume of 2× Laemmli sample buffer (Biorad) supplemented with 5% β‐mercaptoethanol. Samples were denaturated at 95°C for 5 min and then separated on 10% Tris‐HCl polyacrylamide gels (Biorad) and transferred to PVDF membranes (Invitrogen). Membranes were blocked in 5% milk in Tris‐buffered saline with 0·1% Tween 20 and then incubated with rabbit anti‐NF‐κB (p65; 1 : 200; sc‐372, Santa Cruz Biotechnologies) overnight at 4°C or for 1 h at room temperature, followed by incubation with HRP‐conjugated anti‐rabbit antibodies (1 : 30 000; Biorad) for 1 h at room temperature. Signals were detected with the SuperSignal West Pico Chemiluminescent kit (Thermo Scientific). Band intensities were quantified using ImageJ software. The degree of nuclear translocation was calculated as the ratio between nuclear and cytosolic NF‐κB. For each experiment, results were expressed in relation to control cells.