Mouse anti p65

For immunoblot analysis, cells were lysed in a cell lysis buffer containing 50 mM Tris, pH 8, 150 mM NaCl, 1 mM EDTA, 10% (vol/vol) glycerol, 1% (vol/vol) Triton X-100, and 0.05% (vol/vol) NP-40 supplemented with protease inhibitors (Roche). Proteins were then quantified using a bicinchoninic acid protein assay kit (Pierce). For immunoblotting of phosphorylated proteins, the cell lysis buffer was also supplemented with phosSTOP phosphatase inhibitor cocktail tablets (Roche), and samples were subjected to SDS-PAGE immediately after lysis. The following primary antibodies were from the indicated sources: mouse anti-α-tubulin, Upstate Biotech; mouse anti-pIκBα (S32/36), Cell Signaling Technology; mouse anti-total IκBα (tIκBα), a kind gift from Ron Hay, University of Dundee, Dundee, United Kingdom; mouse anti-p65, Santa Cruz; and mouse anti-lamin A/C, Abcam. Mouse anti-D8 monoclonal antibody AB1.1 (33 (link)) and the rabbit anti-A49 polyclonal antiserum (24 (link)) have been described previously. Primary antibodies were detected with goat anti-mouse/rabbit IRdye 800CW infrared dye secondary antibodies, and membranes were imaged using an Odyssey infrared imager (LI-COR Biosciences).

WT or GCN2^−/− MEFs were infected with DENV at moi 3 and incubated for indicated time points (as indicated in the results section). Post incubation, the infected cells were fixed with 4% paraformaldehyde in 1X PBS for 15 min followed by permeabilization with 0.2% Triton-X 100 for 20 min at room temperature. The cells were further blocked with 10% FBS or 1% BSA for 1 h at room temperature. Following washes with 1X PBS, cells were incubated with primary Ab for 2 h at 37°C followed by incubation with fluorescent secondary Abs, Alexa Fluor-488 (Invitrogen) at 37°C for 1 h. Next, cells were washed three times with 1X PBS and immunostained cells were finally mounted using Prolonged Gold Anti-fade Reagent containing DAPI (Invitrogen) or Vectashield Mounting Medium with DAPI (Vector Laboratories). The primary antibodies used were goat anti-COX2 (Santa Cruz), mouse anti-Dengue 1, 2, 3, 4 (GeneTex), mouse anti dsRNA-J2 (English and Scientific Consulting Kft.), and mouse anti-p65 (Santa Cruz). The immunofluorescence images were captured under 63X oil based objective using a LSM510 confocal microscope (Zeiss). Further, the images were processed and analyzed in Zeiss LSM5 image browser. The analysis of intensity profiles was carried out using Fiji/Image J software (NIH).

Equal amounts of total protein samples from the hippocampus were separated via SDS/PAGE (10% Bis‐Tris gel; Invitrogen), following which they were transferred onto a PVDF membrane (Bio‐Rad Laboratories) and blocked in TBST buffer (25 mM Tris‐HCl, 160 mM sodium chloride, and 0.05% Tween‐20) containing 5% (wt/vol) bovine serum albumin (Santa Cruz Biotechnology) for 1 hr at 25°C. The membrane was then incubated with primary antibodies overnight at 4°C. This was followed by incubation with the corresponding secondary antibody for 1 hr at room temperature. Primary antibodies included mouse anti–cleaved caspase‐3 (1:400, Cell Signaling Technology), mouse anti‐Bax (1:1000, Santa Cruz Biotechnology), mouse anti‐Bcl‐2 (1:500, Santa Cruz Biotechnology), mouse anti‐p65 (1:500, Santa Cruz Biotechnology), and rabbit anti‐GAPDH (1:600, Cell signaling Technology). Immunoblot bands were visualized using an ECL Western blot kit (NeoBioscience Biotechnology, China). An Eastman Kodak Image Analyzer (Rochester, NY) was used for semiquantification of the protein signal intensity.

Equal volumes of cell lysates were separated by NuPage ® Novex 4–12% Bis-Tris Gels (Life Technologies) in MOPS SDS running buffer (Life Technologies) and transferred onto nitrocellulose membranes (Sigma-Aldrich). Membranes were probed with primary antibodies (rabbit anti-p50, Santa Cruz, 1:1000 dilution or mouse anti-p65, Santa Cruz, 1:1000 dilution) and signal detection was determined using the Immobilon® Western HRP Substrate (Milipore) on the Syngene G:Box chemi XL gel documentation system. Densitometry was analysed using GeneTools software on the same system.

Whole-cell extracts were prepared and immunoblots performed on 30 µg of extract using standard procedures (Thoms et al., 2010 (link)). Primary antibodies were: rabbit anti-p65 (C-20) (Santa Cruz), mouse anti-p65 (Santa Cruz Biotechnology), rabbit anti-p65 (acetyl K310) (Abcam), mouse anti-His (generated in house), mouse anti-p300 (BD Pharmingen), rabbit anti-acetylated lysine (Cell Signaling), mouse anti-COMMD1 (Abnova), anti-XIAP (Santa Cruz) and anti-GST–HRP (Abcam) antibodies. Actin (Amersham) controlled for protein loading.

MCF-7/HER2-5 cells were seeded in glass coverslips inside the 6-cm dish and cultured to 80% confluence. The cells were then treated with compounds, either alone or in combination with doxorubicin, and incubated for 24 h. After 24 h, the cells were fixed using 70% ethanol and incubated for 15 min at room temperature. After rinsing with PBS, the cells were incubated with the 1% BSA as blocking serum for 30 min at room temperature. Then, the cells were incubated with a primary antibody [mouse anti-p65 (Santa Cruz) or mouse anti-HER2 (Santa Cruz)] overnight at 4°C followed by secondary antibody (Santa Cruz) for 1 h. Then, DAPI staining was used to label nuclei for 10 min at room temperature in the dark. The cells were again rinsed with PBS and placed on sliding glass. The slides were analyzed and photographed using a fluorescence microscope.