To detect the putative suppressive effect of BAT or taurine on the nuclear translocation of phosphorylated NF-κB p65 protein (Ser 536), immunofluorescence experiments were performed. In detail, 24-well plates were seeded overnight with 4.5x104 cells/well. The cells were pre-treated with 0.1-0.5 mM BAT or 100 mM taurine diluted in pure DMEM without FBS for 1.5 h at 37°C. Subsequently, LPS (100 ng/ml) diluted in full DMEM was added to each well, and the cells were incubated for 24 h at 37°C, as previously described (38). The Mφs were washed with 1X phosphate-buffered saline (PBS) for 5 min and fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min. The fixed cells were permeabilized with 1x PBS/0.5% Triton X-100 for 5 min at 4°C and blocked with 1% BSA/1X PBS, diluted in 1X PBS at room temperature. The Mφs were then incubated with the primary anti-phospho-NF-κB-p65 monoclonal antibody (mAb; #3033; Cell Signaling Technology, Inc.) diluted in 1% BSA/1X PBS at 4°C overnight. The following day, the cells were incubated with (4 μg/ml) Alexa Fluor 488-labeled secondary antibody (A11008; Thermo Fisher Scientific, Inc.) diluted in 1% BSA/1X PBS for 1 h at room temperature, after washing with 1X PBS. Hoechst dye No. 33342 (B2261; Sigma-Aldrich Merck KGaA; 0.5 μg/ml) was used for cellular chromatin staining. Finally, the coverslips were mounted in Prolong Gold antifade media (Molecular Probes, Inc.) and the cells were observed under a confocal microscope (Leica Microsystems GmbH) with an excitation wavelength of 355 nm for Hoechst and with an excitation wavelength of 488 nm for phosphorylated NF-κB (Ser 536). The LAS AF program was used to acquire the images. Experiments were repeated independently 3 times and representative images are presented.

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