Mouse monoclonal anti p65 antibody

After fixation, cells were permeabilized in 0.1% Triton X-100 for 10 minutes, incubated in PBS supplemented with 0.5% BSA for 2 hours and then overnight at 4°C with different combinations of primary antibodies. NF-κB p65 localization was visualized by using a mouse monoclonal anti-p65 antibody (Santa Cruz Biotechnology, USA), TIFA was visualized with a polyclonal rabbit anti-TIFA primary antibody (Sigma-Aldrich), and LAMP1 was visualized with an anti-mouse anti-LAMP1 (Abcam). Cells were then stained with Alexa 647- or Alexa 488-conjugated secondary antibodies (Invitrogen, Carlsbad, USA). DNA and F-actin were stained with Hoechst and FITC-phalloidin, respectively. The production of IL-8 was measured by immunofluorescence using an anti-human IL-8 antibody in 0.2% saponin in PBS (BD Pharmingen, San Jose, USA) 4 hours post infection.

Nuclear proteins (including NF-κB p65) were isolated and analyzed by Western blot. The cells were washed twice with ice-cold PBS and suspended in NE buffer A [10 mM Hepes-NaOH (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, proteinase inhibitor, 1 mM DTT, and 1 mM PMSF]. Intact nuclei were released from the cells by several washes with NE buffer B [NE Buffer A supplemented with 0.3% NP-40]. Nuclear membranes were damaged by adding NE buffer C [12.5% glycerol, 1mMTris-HCl (pH 6.5), 0.1 mM EDTA], followed by three cycles of sonication. A mouse monoclonal anti-p65 antibody (1:2,000, Santa Cruz, USA) was used to analyze the translocation of NF-κB to nuclei by standard Western blot analysis as described above. A rabbit polyclonal anti-histone H3 CT pan antibody (1:5,000, Upstate, USA) and a mouse monoclonal anti-α-tubulin antibody (1:10,000, Sigma, USA) were used as loading controls for nuclear and cytoplasmic proteins, respectively.

Paraffin-embedded tissue sections (5 mm) mounted onto glass slides were hydrated in xylene, graded alcohol. Antigen retrieval was performed by microwave (5 min at 700 W) using a sodium citrate buffer (10 mM, pH 6.0). Endogenous peroxidase activity was quenched with 3% H₂O₂. Nonspecific binding was blocked with swine serum diluted 1 : 10 in BSA 1%. Immunostaining was performed using the mouse monoclonal anti-p65 antibody dilution 1 : 50 in BSA 1% (Santa Cruz Biotechnology, Dallas, TX) and incubated for 1 h at room temperature. From the secondary antibody to the chromogen reaction, a Universal LSAb2 HRP kit (DakoCytomation, Glostrup, DK) was used according to the manufacturer's instruction as previously described [20 (link)].