Visualizing NF-κB Translocation in HeLa Cells

HeLa cells were transfected with EV or plasmids encoding N-terminal TAP-tagged B14, C2, C2-B (aa 1−212), C2-K (aa 213−512) or F3 using the LT1 transfection reagent (MirusBio). After 24 h cells were starved in DMEM without serum for 3 h and then stimulated with 40 ng ml⁻¹ TNFα for 30 min. Cells were washed three times in ice-cold PBS and processed for immunofluorescence staining and imaging as described [47 (link)]. Polyclonal rabbit anti-Flag (F7425, Sigma-Aldrich) and mouse monoclonal anti-p65 clone F-6 (sc-8008; Santa Cruz) were used as the primary antibodies and goat anti-rabbit 546 and donkey anti-mouse 488 were used as the secondary antibodies (Jackson Immunoresearch). Images were analysed using the Zeiss Zen microscope software and ImageJ. Experiments were performed in triplicate and carried out three times. For each repeat, 100 Flag-positive cells were analysed for each condition and the percentage of cells showing nuclear p65 determined.