Primary antibody against p65

The cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with methanol at −20°C for 10 min. After blocking with 4% bovine serum albumin, the primary antibody against p65 (Cell Signaling) was applied at a 1/400 dilution for 1 h. Secondary antibody (Goat Anti-Rabbit IgG H&L [Alexa Fluor 488]; Abcam, Cambridge, UK) was applied at a 1/1,000 dilution for 30 min with 10 μg/mL propidium iodide (Sigma-Aldrich). After mounting with Fluoro-KEEPER Antifade Reagent (Nacalai Tesque), subcellular localization of p65 and nucleus was visualized using a Nikon BioStation IM (NIKON, Tokyo, Japan) with FL1 and FL2 detectors, respectively. The ratio of nuclear p65-positive cells to total cells in the fields was calculated.

The frozen livers were lysed using RIPA buffer (Beyotime Shanghai, China) containing protease inhibitors, incubated on ice for 30 min, and centrifugation at 12,000× rpm for 5 min. The protein levels in the supernatant were determined using the BCA protein assay kit (Beyotime Shanghai, China). Subsequently, equal amounts of proteins (20 μg) were separated on 10% SDS-PAGE and transferred to PVDF membranes (Millipore corp., Billerica, MA, USA). The membranes were blocked with 5% skim milk for 1 h and incubated overnight with primary antibody against p65 (#6956; 1:1000; Cell Signaling, Danvers, MA, USA), IκBα (#4814; 1:1000; Cell Signaling, Danvers, MA, USA), and β-actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C. Then, the membranes were incubated with HRP-conjugated secondary antibodies (3:5000; Beyotime Shanghai, China) for 30 min at room temperature. The protein expression signals were visualized by ECL (Beyotime Shanghai, China), and band intensity was quantified using the Image J software (NIH, Bethesda, MD, USA).

Firstly, cells that grew on glass coverslips were fixed and then permeabilized with 0.25% Triton X-100 for 10 min, blocked with 0.8% BSA for 1 h at room temperature. Next, cells were incubated with the primary antibody against p65 (Cell Signaling Technology) overnight at 4 °C and treated with appropriate secondary antibody. The nucleus was counterstained with DAPI. The experiment was performed as described previously [20 (link), 21 (link)].

MDA-MB-231 cells were plated and cultured on poly-d-lysine-coated glass coverslips in six-well plates. Twenty-four hours after transfection, the cells were treated with TNF-α (GenScript, NJ, USA) for 15 and 30 min. Then the cells were washed with cold PBS three times and fixed with 4% formaldehyde (Thermo Fisher, MA, USA) in PBS for 15 min at room temperature. They were then rinsed with cold PBS twice and permeabilized with PBS containing 0.1% Triton X-100 (Sigma) for 10 min at room temperature followed by washing with PBS three times. To block the nonspecific binding of the antibodies, samples were incubated with 3% BSA (Invitrogen) in PBS for 1 h at room temperature. Primary antibody against p65 (Cell Signaling Technology) was added to the samples and incubated for overnight at 4 °C. After washing with PBS three times, the cells were incubated with anti-rabbit secondary antibody conjugated with Alexa 488 (Invitrogen) for 1 h at room temperature followed by three times of PBS wash. Cells were stained with a DAPI (1 ng/ml; Sigma) for 5 min. Images were obtained with an LSM700 Confocal microscope (Carl Zeiss, Germany).

We seeded hDPCs into 24-well plates. Then, we exposed them to 5 μg/mL LPS with or without 5 μg/mL bromelain or Bay11-7082 (Sigma-Aldrich), an inhibitor of NF-κB, for 3 h. Then, we fixed the cells with 4% paraformaldehyde for 10 min, permeabilized them with 0.1% Triton X-100 for 15 min, and blocked them with 3% BSA for 1 h. We incubated the cells with a primary antibody against p65 (1:500; Cell Signaling) overnight at 4 °C and then incubated them with an antirabbit secondary antibody (1:200; Cell Signaling) for 1 h at room temperature. Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and stored at 4 °C in the dark. Then, we observed the cells under a confocal laser scanning microscope (CLSM) (Carl Zeiss, Oberkochen, Germany).