Anti phosphorylated c fos

Cells were suspended in ice-cold RIPA buffer, and cell lysate proteins (100 μg) were electrophoresed on SDS polyacrylamide gel and then transferred to a nitrocellulose membrane. The blots were blocked for 1 h at room temperature in a blocking solution (5% non-fat dry milk in Tris buffered saline containing 0.05% Tween-20; TBST), and then immersed in primary antibody (4 °C, overnight, shaking). Immunoreactive signals were generated in a luminescence detection system (Amersham, Franklin Lakes, NJ, USA) using horseradish peroxidase labeled secondary antibody (Cell Signaling Technology, Danvers, MA, USA). The primary antibodies used were as follows: anti-RONβ was from Santa Cruz Biotechnology, and anti-phosphospecific Erk-1/2, anti-phosphospecific JNK anti-phosphospecific p38 MAPK, anti-Egr-1, anti-phosphorylated c-jun, anti-phosphorylated c-fos, anti-IκBα, anti-phosphorylated IκBα (Ser32), and anti-phosphorylated NF-κB p65 antibodies were purchased from Cell Signaling Technology. To measure the loading quantity of samples, the blotted membrane was stripped with 62.5 mM Tris−HCl (pH 7.4) containing 2% SDS and 100 mM 2-mercaptoethanol, followed by hybridization with β-actin antibody (Santa Cruz, Dallas, Texas, USA) or antibodies against total Erk-1/2, JNK and p38MAPK (Cell Signaling Technology, Danvers, MA, USA).

The following reagents were used: dexamethasone (Sigma, Dorset, UK), H₂O₂ (Sigma, Dorset, UK), azithromycin, LY294002, BEZ235 (Selleck, Houston, USA), and tumor necrosis factor α (TNFα) (Sigma, Dorset, UK). The following antibodies were used: goat anti-rabbit (Abcam, Cambridge, UK), anti-phosphorylated NF-κB (Cell Signaling Technology, Danvers, USA), anti-phosphorylated c-Jun (Cell Signaling Technology, Danvers, USA), and anti-phosphorylated c-Fos (Cell Signaling Technology, Danvers, USA).