Mouse anti p65 antibody

To monitor p65 translocation, J774A.1 cells were treated with compounds for 1 h, followed by LPS (100 ng/ml) treatment for 30 min. The cells were fixed with 4% paraformaldehyde (Sigma; #158127) in PBS (w/v) for 30 min at room temperature. After washing with 1× PBS (Welgene; ML008-02) three times, the fixed cells were permeabilized with PBS containing 0.1% Triton X-100 for 10 min. The cells were washed with 1× PBS three times and then blocked with 4% bovine serum albumin (MP Biomedicals; #0216006980) in PBS (w/v) for 1 h. The cells were incubated with mouse anti-p65 antibody (Santa Cruz; sc-8008, 1:200 diluted in 1% BSA–PBS) at 4 °C overnight and washed with PBS three times. FITC-labeled goat anti-mouse IgG antibody (Abcam; ab6785, 1:200 diluted in 1% BSA–PBS) was added to the sample and incubated for 1 h at room temperature. The remaining antibodies were finally washed three times with PBS. Nuclei were stained with Hoechst 33342 (Thermo; #62249). The cellular p65 proteins were imaged by fluorescence microscopy (GE Healthcare; Delta Vision) and quantified by the line profiling method.

For immunofluorescence microscopy, A549 cells were seeded into six-well plates containing sterile glass coverslips. Following infection with vv811 recombinant viruses and stimulation with TNF-α or IL-1β, the cells were washed twice with ice-cold PBS and fixed in 4% (vol/vol) paraformaldehyde. The cells were then quenched in 150 mM ammonium chloride, permeabilized in 0.1% (vol/vol) Triton X-100 in PBS, and blocked for 30 min in 5% (vol/vol) FBS in PBS. The cells were stained with mouse anti-p65 antibody (Santa Cruz) for 1 h, followed by incubation with donkey anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen Molecular Probes). Coverslips were mounted in Mowiol 4-88 (Calbiochem) containing DAPI (4′,6-diamidino-2-phenylindole). Images were taken on a Zeiss LSM780 confocal microscope using Zen2011 acquisition software.

Cells were seeded into 6-well plates containing sterile glass coverslips. Following stimulation with IL-1β (25 ng/ml) for 30 min, the cells were washed twice with ice-cold PBS and fixed in 4% (w/v) paraformaldehyde. The cells were then quenched in 150 mM ammonium chloride, permeabilized in 0.1% (v/v) Triton X-100 in PBS, and blocked for 30 min in 5% (v/v) FBS in PBS. The cells were stained with rabbit anti-FLAG (Sigma, 1:300) and mouse anti-p65 antibody (Santa Cruz, 1:50) for 1 h, followed by incubation with goat anti-rabbit IgG Alexa Fluor 488 and goat anti-mouse IgG Alexa Fluor 568 secondary antibodies (Invitrogen). Coverslips were mounted in Mowiol 4-88 (Calbiochem) containing DAPI (4′,6-diamidino-2-phenylindole). Images were taken on a LSM 510 META confocal laser scanning microscope (Zeiss) using the LSM image browser software (Zeiss).