F0514

The following antibodies were used: anti-VP16 monoclonal antibody (ab110226; Abcam), anti-gB monoclonal antibody (ab6505; Abcam), anti-β-actin monoclonal antibody (Sigma-Aldrich), anti-P65 polyclonal antibody (F0514; Santa Cruz), secondary antibody (Sigma-Aldrich), anti-phospho-IκB-α monoclonal antibody (9246; Cell Signaling), anti-Phospho-P65 monoclonal antibody (3033p; Cell Signaling), anti-phospho-JNK monoclonal antibody (9251; cell signaling), anti-ICP6 polyclonal antibody was generated in rabbit by immunization with recombinant ICP6 N-terminal polypeptide. Secondary antibody binding to Alexa Fluor 488 was purchased from Life Technologies.

Cells were cultured in 8-well glass (Merk Millipore, Cork, COR, Ireland). After been stimulated, cells were fixed for 30 min with 4% paraformaldehyde at 4 °C, washed in PBS and permeabilized with a PBS solution containing 0.5% Triton X-100 for 10 min, followed by another incubation period of 30 min in PBS with 0.5% Triton and 0.2% BSA at room temperature. Then, cells were incubated with a rabbit polyclonal anti-p65 antibody (#F0514, Santa Cruz Biotechnology) for 2 h at room temperature, washed with PBS with 0.5% Triton and incubated with Alexa Fluor 568-conjugated donkey anti-rabbit IgG (#A10042, Invitrogen) for 1 h at room temperature. The cells were then washed and counterstained with DAPI. Images were taken using an Olympus BX61 microscope (Olympus, Tokyo, Japan) and further analyzed with ImageJ software version 1.50 e (National Institutes of Health, Bethesda, WA, USA). Fluorescence intensity of the red channels across individual cells (n = 30 per condition) was obtained using 200 × 50 px transects. To obtain plot profiles, a baseline correction was applied using GraphPad Prism 5.01 software, which defined the baseline as the average of the first 15 and last 15 values of the transect, and performed the calculation as the difference between the original value and baseline.