Rabbit anti phospho nf κb p65 antibodies

After treatment, hPDLSCs were washed with cold PBS and lysed in RIPA lysis buffer with the supplied with protease/phosphatase inhibitor cocktail. Protein concentrations were measured by BCA protein assay kits. Total protein (25 μg) was fractionated with SDS-PAGE gels and transferred onto PVDF membranes. The PVDF membranes were blocked with 5% skim milk and incubated with primary antibodies at 4°C overnight. Rabbit anti-Bcl2 antibodies (1: 1000), rabbit anti-Bax antibodies (1: 1000), rabbit anti-Phospho-cleaved caspase3 antibodies (1: 500), rabbit anti- caspase3 antibodies (1: 1000), rabbit anti-bmp2 antibodies (1: 1000), rabbit anti-Oct4 antibodies (1: 1000), rabbit anti-Sox2 antibodies (1: 1000), rabbit anti- Runx2 antibodies (1: 1000), rabbit anti-MyD88 antibodies (1: 1000), rabbit anti-NF-κB p65 antibodies (1: 500), rabbit anti-Phospho-NF-κB p65 antibodies (1: 1000), rabbit anti-TLR4 antibodies (1: 1000), and rabbit anti- GAPDH antibodies (1: 1000) were obtained from Cell Signaling Technology. After washing 3 times with PBS, appropriate secondary antibodies were applied for 2 h, and signals were analyzed using the Tanon-5200 Chemiluminescence Imager with enhanced chemiluminescence Western blotting substrate (Millipore, Billerica, MA).

The skin samples were crushed in liquid nitrogen and solubilized at 4°C in lysis buffer (0.5% sodium deoxycholate, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 100 μg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and protease inhibitor cocktail). Proteins (30 μg) were separated on SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). Non-specific protein binding was blocked by incubating the membranes in 5% w/v non-fat milk powder in TBS-T (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, and 0.1% v/v Tween-20), and then the membranes were incubated with rabbit anti-phospho-NF-κB p65 antibodies or rabbit anti-NF-κB p65 antibodies (Cell Signaling Technology) diluted 1:1,000 overnight at 4°C or with mouse anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:10,000 for 30 minutes at room temperature. The membranes were then washed 3 times in TBS-T for 5 minutes each and finally incubated with either HRP-conjugated anti-mouse or anti-rabbit antibodies at a dilution of 1:10,000 for 60 minutes at room temperature. Protein bands were detected using the ECL Plus kit (GE Healthcare, Buckinghamshire, UK), and the intensity of the bands was quantified using the NIH Image J software program.