Nf κb p65 c22b4 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.

Male C57BL/6J mice, 6–8 weeks old, were obtained from the Animal Research Committee of the Institute of Biology and Cell Biology (Shanghai, China) and housed in a specific environment as previously described 21, 22. All animal experiments were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of the Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong Province, China. LPS (Escherichia coli, 055:B5) and d‐GalN were obtained from Sigma‐Aldrich (St Louis, MO, USA; catalog no.: L2880 and G0500). Poly(I:C) was from InvivoGen (San Diego, CA, USA; catalog no.: tlrl‐picw). Mouse mAb to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; catalog no.: 60004‐1‐lg), β‐actin (catalog no.: 60008‐1‐lg) and lamin B1 (catalog no.: 66095‐1‐lg) were purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Anti‐MuRF2 (ab3) antibody produced in rabbit was purchased from Sigma‐Aldrich (catalog no.: SAB2102565). MuRF2 (C‐20) antibody was acquired from Santa Cruz Biotechnology (Dallas, TX, USA; catalog no.: sc‐49454); NF‐κB p65 (C22B4) rabbit mAb was obtained from Cell Signaling Technology (Danvers, MA, USA; catalog no.: 4764).