Hela shCnt and HeLa shMyD88 cells were activated with 20 ng/mL IL-1β or 10 ng/mL TNFα (ProSpec, Rehovot, Israel) for 30 min or 1 h, respectively. Cells were then fixed (3.7% PFA in PBS for 10 min), permeabilized (0.25% Triton-X100) and blocked (2% BSA in TBS) at 4°C for 16 h. Cells were then stained with 0.6 µg/mL rabbit anti-p65 in 2% BSA in TBS (Santa Cruz Biotechnology, Dallas, TX, USA) followed by 0.5 µg/mL CY-3 goat anti-rabbit antibody (Jackson ImmunoResearch, Baltimore Pike, PA, USA). Cytoplasmic vs. nuclear localization was analyzed by fluorescent microscopy (15 (link)) (Nikon-Ti microscope).
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