Total iκbα 44d4 rabbit mab

Polarized tonsil epithelial cells were incubated with HIV tat and/or gp120 recombinant proteins or cell-free HIV virions with or without MAPK inhibitor U0126 at 20 µM. After 5 days, cells were lysed and proteins were separated and transferred to nitrocellulose membranes (GE Healthcare). MAPK activity was measured by detection of phosphorylated and total ERK1/2 protein using phospho-p42/44 MAPK (Erk1/2) (Cell Signaling Technology) and total p42/44 MAPK (Erk1/2) (Cell Signaling Technology). To determine NF-κB activity, we used phospho-Iκbα (Ser32) (14D4) (Cell Signaling Technology), total Iκbα (44D4) rabbit mAb (Cell Signaling Technology), phospho-NF-κB p65 (Se536) (93H1) rabbit mAb (Cell Signaling Technology) and NF-κB p65 (C22B4) rabbit mAb (Cell Signaling Technology). MMP-9 expression was detected using mouse antibody to MMP-9 (Cell Signaling Technology). Protein bands were visualized using the ECL detection system (GE Healthcare). For quantitative analysis of protein bands, films were scanned and saved in TIFF format. The integrated density (pixel intensity over a selected area) of protein bands was quantified by ImageJ with background correction.

Media and serum were purchased from Invitrogen (Paisley, UK). Antibodies were sourced as follows: FADD mouse mAb (610400) was from BD Transduction Laboratories (San Jose, CA, USA); GST tag mouse mAb (71097) was from Novagen (Watford, UK); cleaved caspase-3 (Asp175) (9661) rabbit polyclonal antibody, cleaved caspase-8 (Asp384) (11G10) mouse mAb, Bid (2002) rabbit polyclonal antibody, Phospho-IκBα (Ser32) (14D4) rabbit mAb and total IκBα (44D4) rabbit mAb were sourced from Cell Signaling Technology Inc. (Hitchin, UK); caspase-8 rabbit polyclonal antibody was generated in our laboratory; caspase-8 mouse mAbs (C15 and N2) were kind gifts from Prof. PH Krammer (German Cancer Research Center, Heidelberg, Germany); the p85α and pan-Phospho-tyrosine (2C8) mouse mAbs were from Santa Cruz Biotechnology, Inc. (Middlesex, UK); Phospho (Tyr380) caspase-8 was a kind gift from Prof. D Barila (University of Rome Tor Vergata, Rome, Italy). Horseradish peroxidase-conjugated secondary antibodies were obtained from Sigma-Aldrich (goat anti-mouse; Dorset, UK) and DAKO (goat anti-rabbit; Cambridge, UK). The caspase-8 substrate, Ac-Ile-Glu-Thr-Asp-amino-4-trifluoromethyl coumarin (Ac-IETD.AFC) was purchased from MP Biomedicals (Cambridge, UK). All other chemicals were of analytical grade and obtained from Sigma-Aldrich or Fisher (Loughborough, UK).