Phosphorylated ikk

For each Western blot analysis, 2×10⁵ cells were seeded. When subconfluence reached 80%, RA FLSs were pretreated with DMSO (5%) or NH125 for 24 h or transfected with eEF2K siRNA or si-C. Then, RA FLSs were stimulated with or without TNF-α (10 ng/mL) for 15 min. Proteins in RA FLSs were separated by SDS-PAGE (12%) and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were incubated for 12 h at 4° C with the following primary antibodies: phosphorylated-IKK, IKK, IκBα, phosphorylated-IκBα, phosphorylated-AKT, AKT and β-actin (1:1000 dilution, all from Cell Signaling Technology, Danvers, MA, USA). Then, the PVDF membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1: 5000 dilution, Cell Signaling Technology) at room temperature for 1 h. The protein bands were visualized with chemiluminescence (Millipore, Boston, MA, USA). The membrane was scanned and densitometry quantified by ImageJ analysis system (National Institutes of Health). The intensity of each protein was First normalized to β-actin bands and represented as a ratio of controls.

The method of Western blotting analyses was performed as described previously.17 (link) Bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA) was used to detect protein concentrations. Equal amounts of protein were solubilized in Laemmli buffer (pH 6.8, 62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.0625‰bromophenol blue), boiled for 5 minutes, separated by 12% SDS-PAGE, and transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were probed with the following primary antibodies: phosphorylated-IKK, IκBα, phosphorylated-IκBα and β-actin (all from Cell Signaling Technology, Danvers, MA, USA) in TBS/Tween-20 containing 5% non-fat milk at 4°C overnight. The membranes were incubated with the secondary antibodies for 1 hr at room temperature. A peroxidase-conjugated secondary antibody (1:5000 dilution, BOSTER Biological Technology Co. Ltd, Wuhan, China) and enhanced chemiluminescence Western blot detection reagents (NCI4106, Pierce, ThermoFisher) were used to visualize the target proteins. Each blot is representative of at least 3 independent experiments. The film was scanned by a high-resolution scanner, densitometry was performed using AlphaEaseFc software on a Fluorchem 8900 system (Alpha Innotech, San Leandro, CA, USA).