Rabbit anti nf κb p65 igg

MG6 cells were treated with Pg LPS (10 μg/mL) for 3 h in the absence and presence of hBD3 (1 μM) and were fixed with 4% paraformaldeyte. They were then incubated with rabbit anti-NF-κB p65 IgG (Abcam). After washing with PBS, cells were incubated with donkey anti-rabbit Alexa 555 (ThermoFisher Scientific, Waltham, MA, USA), then incubated with Hoechst and mounted in Vectashield anti fading medium (Vector Laboratories, Newark, CA, USA). Fluorescent images were taken using a confocal laser-scanning microscope (FV1000). The line plot profile was analyzed using Image J.

Total protein from the cells was extracted using radio-immunoprecipitation lysis buffer containing 60 lg/ml phenylmethylsulfonyl fluoride [Tiangen Biotech (Beijing) Co., Ltd., Beijing, China]. The protein concentration was determined by Bradford assay (Sigma-Aldrich). Western blot analysis was conducted using the following primary monoclonal anti-human antibodies: Rabbit anti-Aurora-B IgG, (1:200), rabbit anti-PI3K IgG (1:1,000), rabbit anti-phosphorylated (p)Akt IgG (1:800) goat anti-Akt IgG (1:1,000), rabbit anti-NF-κB (p65) IgG (1:400), rabbit anti-matrix metalloproteinase (MMP)-2 (1:1,000) and rabbit anti-MMP-9 IgG (1:1,000), which were all purchased from Abcam and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (1:5,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and the corresponding mouse anti-rabbit, mouse anti-goat and goat anti-mouse monoclonal secondary antibodies (ZSGB-BIO, Beijing, China). The immune complexes were detected using the pro-light Streptavidin-HRP kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Six independent experiments were performed over numerous days.