NF-κB p65 Translocation in Macrophages

Macrophages were grown on coverslips in 12-well plates and pretreated for 1 h with 7.5 μM curcumin, GG6, GG9, or 1 μM CLI-095 and then stimulated with 100 ng/ml Ultrapure LPS-EB for an additional 90 min. Cells were fixed with 4% paraformaldehyde (pH 7.4, for 15 min at room temperature) and subsequently blocked with 5% normal goat serum/0.1% Triton X-100 in PBS for 1 h as described [30 (link)]. Cells were incubated with a mouse anti-p65 antibody (NF-κB p65, 1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by an Alexa Fluor 555-conjugated anti-mouse secondary antibody (1 : 1000; Invitrogen). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, Sigma), and coverslips were mounted on microscope slides with Fluoromount-G mounting medium (Thermo Fisher Scientific, Milan, Italy). Fluorescent images for p65 staining were captured with a confocal laser-scanning microscope (Zeiss LSM 800; Carl Zeiss AG, Oberkochen, Germany) and analyzed with ZEN 2.0 imaging software (Carl Zeiss AG).