Anti nf κb p65

The following phospho-specific monoclonal antibodies were used in 3 different panels during the flow cytometry protocol described previously (17 (link)): Alexa Fluor®647 conjugated anti-STAT4 (pY693, clone 38/p-STAT4, panel 1), anti-STAT 1 (pS727, clone K51-856, panel 2), and anti-STAT3 (pS727, clone 49/p-STAT3, panel 3); PerCP-Cy^TM 5.5 conjugated anti-ERK1/2 (pT202/pY204, clone 20A, panel 1), anti-STAT1 (pY701, clone 4a, panel 2), and anti-STAT3 (pY705, clone 4/P-STAT3, panel 3); and PE-Cy^TM7 conjugated anti-p38 MAPK (pT180/pY182, clone 36/p38, panel 2), and anti NF-κB p65 (pS529, clone K10-895.12.50, panel 1), anti-STAT5 (pY694, clone 47/STAT5(pY694), panel 3) (all from BD Biosciences, San Jose, CA, USA). Cell surface markers incorporated in the assays were BV786 conjugated anti-CD3 (clone SK7, BD Horizon^TM), Alexa Fluor® 488 conjugated anti-CD20 (clone H1 (FB1), BD Biosciences) and PE conjugated anti-CD56 (clone N901, Beckmann Coulter, CA, USA).

Chromatin immunoprecipitation was performed essentially as described [38 (link)]. Protein-DNA complexes were immunoprecipitated using anti-NF-κB p65 (BD Biosciences) or control IgG. Primer sequences for the binding sites are listed in Additional file 1: Table S1.

Immunostaining was performed on RAW264.7 cells following antigen retrieval using Retrievagen A (Zymed Laboratories, Inc., South San Francisco, CA, USA) at 100°C for 20 min, and endogenous peroxidases were quenched with 3% H₂O₂. RAW264.7 cells were blocked with 2% bovine serum albumin in phosphate-buffered saline, which was followed by staining with primary anti-NF-κB p65 (BD Pharmingen, San Jose, CA, USA) antibodies at room temperature for 1 h. RAW264.7 cells were washed and incubated with secondary antibodies (R&D Systems, Minneapolis, MN, USA), and developed using VECTASTAIN ABC (Vector Laboratories, Inc., Burlingame, CA, USA) and 3,3′-diaminobenzidine (Vector Laboratories, Inc.). Image-Pro Plus analysis software (Media Cybernetics, Inc., Rockville, MD, USA) was used to calculate the NF-κB p65-positive expression in RAW264.7 cells, which was expressed as positive units.

The expression of AT₁R, NF-κB p65 and phosphorylation of IKKα/β, IκBα, oxidative stress markers (NOX2, iNOS), and GAPDH in the renal cortex were determined by Western blotting. The renal cortices were homogenized in ice-cold lysis buffer (PBS with 1 % NP40, 1 mmol/L EDTA, 1 mmol/L PMSF, 10 μg /ml leupeptin and 10 μg/ml aprotinin inhibitor). Equal amounts of total extracted proteins (50 μg) were separated on SDS-PAGE and were transferred onto nitrocellulose membranes (Amersham Life Science, Arlington, TX). The blots were subjected to immunoblot analyses with the primary polyclonal antibodies for rabbit anti-AT₁R, anti-IKKα/β, phospho-IKKα/β, anti-IκBα, phospho-IκBα, NOX2 and iNOS (1:300; Santa Cruz Biotechnology, Santa Cruz, CA), anti-NF-κB p65 (1:400; BD Transduction Laboratory, Minneapolis, MN, USA), anti-Histone and anti-GAPDH (1:500, Santa Cruz Biotechnology). Immunodetection was accomplished by incubating the blots in horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:10,000 dilution). The bands were visualized using enhanced chemiluminescece kit (Amersham, Arlington, TX), and the band intensities were quantified by densitometry using Quantity-One software (Bio-Rad, Hercules, CA), and normalized with GAPDH expression.