Anti c fos

Dingxian pill was prepared in accordance with standards formulated from the Jiangsu Hospital of Traditional Chinese Medicine (Nanjing, Jiangsu Province, China). Pentrazol (PTZ) and pentobarbital sodium salt were purchased from Sigma Aldrich Co. (St. Louis, MO, USA), sodium valproate (VPA) from Sanofi-Aventis Co. (Paris, France), and anti-c-Fos from Novus Biologicals (NB110-75039, Colorado, USA). The SABC-POD kit, DAB coloring kit, and IgG-Biotin were obtained from Boster Company (Wuhan, China). The kits of RT-PCR and Trizol were purchased from Novizan Biotechnology Co. (Nanjing, China).

Primary human arterial endothelial cells and PASMC were lysed in RIPA buffer (Sigma-Aldrich) and 10 μg of protein were run on a 10% SDS polyacrylamide gel, followed by electrotransfer to an Amersham Hybond P 0.45 PVDF Blotting Membrane (GE Healthcare, Wien, Austria). After blocking with 5% non-fat dry milk in TBS-T buffer (Tris-buffered saline with 0.1% Tween-20), the membrane was incubated overnight at 4°C with one of the following antibodies: anti-p38, anti–phospho-p38 (T180/Y182), anti-c-Jun; anti-phospho c-Jun (S73), anti-phospho Erk1/2 (T202/Y204), anti-Erk1/2, anti-phospho c-Fos (S32) all from Cell Signaling and anti-c-Fos (Novus Biologicals, Cambridge, UK) were used at dilution of 1:1000; and anti-TLR4 (Abcam), anti-RAGE (1:500; Abcam) and rabbit anti–alpha-tubulin (1:5000; Cell Signaling, Boston, MA, USA). After washing, the membranes were incubated with horseradish-peroxidase–labelled secondary antibodies (1:5000, anti-rabbit-HRP, Pierce, Thermo Fisher Scientific, Waltham, MA, USA) and proteins detected with Amersham ECL Prime Western Blotting Detection System (GE Healthcare). Antibodies were removed by incubating the membrane for 15 min. with stripping buffer (RestoreTM PLUS Western Blot Stripping Buffer; Thermo Scientific). Densitometric analysis was performed with ImageJ (National Institutes of Health, USA).