Rabbit anti nf κb p65 antibody

Human neutrophil elastase-treated macrophages stimulated with HK-Spn or LPS were fixed and permeabilized using a cell fixation and permeabilization kit (Thermo Fisher Scientific) according to manufacturer instructions, followed by incubation of the cells in a blocking solution (Thermo Fisher Scientific) for 30 min. Samples were stained with rabbit anti-NF-κB p65 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-toll-like receptor (TLR) 2 antibody (clone TL2.1; Thermo Fisher Scientific), or anti-TLR4 antibody (clone HTA125; Thermo Fisher Scientific) in blocking solution. After overnight incubation at 4°C, secondary AlexaFluor 594-conjugated goat anti-rabbit IgG antibody or AlexaFluor 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific) in blocking buffer was added, followed by a 2-h incubation in the dark. Then, samples were observed using a confocal laser-scanning microscope (Carl Zeiss, Jena, Germany). In addition, samples were observed with fluorescence microscopy using Hybrid cell-count software (Keyence, Osaka, Japan) to calculate fluorescence intensity per cell.

Skin, brain and retina tissues from Ikk2^−/fl/Nestine-Cre^+/− and WT (Ikk2^+/fl) control mice were collected and homogenized in cold immunoprecipitation assay buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 0.2% each of deoxycholate, Triton X-100, and Nonidet P-40) containing 1× complete protease-inhibitor mixture (Roche Diagnostics, Indianapolis, IN). Equal amounts of whole-cell lysates were separated on 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to a nitrocellulose membrane. For immunoblotting, the mouse anti-IKK2 antibody (Biosource AH00362, 1∶100 dilution), and mouse anti-β-actin (1∶1000, cat #A2228, Sigma, St. Louis, MO) were used as primary antibodies. The horseradish peroxidase-conjugated goat-anti-mouse secondary antibody and an enhanced chemiluminescence (ECL) system (Amersham Pharmacia, Piscataway, NJ) were used to visualize the signals. For immunobloting of IκBα and α-tubulin in the ARPE-19 cells pre-treated with TPCA-1 and TNFα, rabbit anti-IκBα antibody (1∶250 dilution, Santa Cruz Biotechnology, Inc, sc-371) and mouse anti-α-tubulin antibody (1∶500 dilution, Sigma, T9026) were used. For thermal injured ARPE-19 cells, rabbit-anti-NF-κB p65 antibody (1∶200 dilution, Santa Cruz Biotechnology, Inc., sc-372) and rabbit-anti-NF-κB p65 (Ser536) antibody (1∶500 dilution, Cell Signaling Technology, Inc., Cat #3033) were used.

Rabbit anti-phospho-IκBα antibody, rabbit anti-IκBα antibody, and rabbit anti-β-actin antibody were obtained from Cell Signaling Technology (Beverly, MA). Rabbit anti-NF-κB p65 antibody, anti-Bcl-2 antibody, and anti-Histon H1 antibody were purchased from Santa Cruz Biotechnology (Lake Placid, NY). Rabbit anti-cyclin D1 antibody, anti-cyclin D2 antibody, anti-cyclin D3 antibody, and anti-cyclin E antibody were purchased from PharMingen (San Diego, CA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). IMD-0354, an IKKβ inhibitor 20 (link)–22 , was kindly provided by the Institute of Medicinal Molecular Design Inc. (Tokyo, Japan). Pemetrexed and cisplatin were purchased from LKT Laboratories, Inc. (St. Paul, MN).