Rabbit anti nf κb

The immunofluorescence method used in this study was described by Pandurangan et al. [23 (link)]. Paraffin-embedded colonic tissue sections with a thickness of 5 μm were deparaffinized in xylene and then rehydrated in a graded series of ethanol solutions. The slides were then blocked with 5% BSA in TBS for 90 min. The sections were then immunostained with rabbit anti-NF-κB (Santa Cruz Biotechnology, CA, USA) and anti-pSTAT3^Y705 antibody (Cell Signaling Technology, CA, USA) diluted 1 : 100 with 5% BSA in TBS and incubated overnight at 4°C. After the sections were washed three times with TBS, the slides were incubated with goat and rabbit DyLight 550 secondary antibody (Thermo Scientific, Rockford, IL, USA) diluted 1 : 200 with TBS and incubated in the dark for 120 min at room temperature. The sections were then washed with TBS and incubated with the nucleus-specific counterstain propidium iodide (Nacalai Tesque, Japan) or 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) to stain the cell nuclei. The slides were mounted in an Ultracruz hard-set mounting medium (Santa Cruz Biotechnology Inc., Dallas, TX, USA), coverslipped, and visualized under a FSX100 fluorescent microscope (Olympus, Tokyo, Japan).

2 × 10⁶ WT and TLR2^−/− mouse peritoneal macrophages were seeded in a well of six-well culture plates with sterile glass coverslips. The cells were stimulated with 1 × 10⁶G. lamblia trophozoites for 0 or 60 min at 37°C and washed twice with sterile PBS. After stimulation, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed three times with sterile PBS. Then the cells were incubated overnight in 100 µl permeabilization/wash buffer (0.1% Triton X-100/2% FBS/0.1% azide/PBS) containing rabbit anti-NF-κB (Santa Cruz) at a 1:100 dilution at 4°C. Cells were then washed and incubated with FITC-conjugated goat anti-rabbit IgG (Boster, Wuhan, China) secondary antibody for 1 h at room temperature. The cells were washed, and the coverslips were stained with DAPI at room temperature for 5 min. NF-κB localization was observed using a Zeiss LSM 710 confocal microscope equipped with a 633, 1.4-NA, oil-immersion objective (Carl Zeiss).

The protocols of the western blot were performed as described by Hanson et al. [26 (link)].
Protein extracts, quantified by a Bradford Protein Assay (Bio-Rad Laboratories, CA, USA), underwent SDS-polyacrylamide gel electrophoresis and were transferred to Immobilon-P membranes. The following antibodies were used: rabbit anti-NF-κB, rabbit anti-βcatenin, goat anti-matrin3, goat anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1 : 500; rabbit anti-cyclin E2, cyclin D1, cyclin B1, p21, Pmyt1, Oct4, and mouse anti-cyclin A1, and SSEA-4 (Cell Signalling Technology, Beverly, MA, USA), mouse anti-tubulin, and mouse anti-sc-35 (Sigma-Aldrich St. Louis, MO, USA), rabbit anti-Nrf2 (Abcam, Cambridge, UK), rabbit anti-Nox4 (Novus Biologicals, CO, USA), and mouse anti-pH2A (Ser139), mouse anti-CD90 and anti-CD105 (Millipore, Billerica, MA, USA) rabbit anti-CD73 (Genetex, Irvine, CA, USA), diluted 1 : 1000; peroxidase-labelled anti-rabbit, mouse, and goat secondary antibodies diluted 1 : 3000 (Pierce Antibodies, Thermo Scientific; Rockford, IL, USA). Ab dilution was performed in TBS-T pH 7.6 containing 3% BSA. The membranes were visualized using Supersignal substrate chemiluminescence detection kit (Pierce, Rockford, IL, USA). Anti-actin antibody was used as control of protein loading.