Rabbit anti phospho smad3

Whole-cell extracts were prepared by scraping cells in ice-cold RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonylfluoride PMSF, and 1 mM Na₃VO₄ supplemented with proteinase inhibitor cocktail from Sigma–Aldrich). The lysate was centrifuged at 12,000 × g for 10 min at 4°C and protein concentration was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA). Proteins were separated by electrophoresis on a 10% stacking Tris-glycine gel and transferred to a nitrocellulose membrane. The primary antibodies were goat anti-hKIM-1 (1:1000), anti-mKIM-1 IgG (1:1000), rabbit anti-phospho Smad3 (1:500), anti-E-cadherin (1:1000, Cell Signaling, Danvers, MA), and mouse anti-β-actin antibody (Sigma–Aldrich). The blots were then washed in PBS-0.3% Tween-20 and incubated with the second antibody (donkey anti-goat, anti-rabbit, or anti-mouse IgG) conjugated to horseradish peroxidase for 90 min at room temperature and washed. Detection was accomplished by enhanced chemiluminescence Western blotting (ECL, Amersham), and blots were exposed to X-ray film (Hyperfilm-ECL, Amersham). Western blots were quantified by densitometry (Scion Image for Windows, Scion Corporation, Fredrick, MD).