Tgif1

Both the treated and untreated HCF cultures were lysed in a radioimmunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, CA) followed by centrifugation at 10,000 g for 10 min. Samples were suspended in a NuPAGE LDS buffer containing a reducing agent (Life Technologies Corporation, Grand Island, NY) and heated at 70 °C for 10 min. Protein samples were resolved by NuPAGE Novex Bis-Tris mini gels (Invitrogen) and transferred onto the polyvinylidene difluoride membranes using wet transfer at 25 V. The transferred proteins were detected by incubating the membrane with primary antibodies: TGIF1, TGIF2 (Santa Cruz Biotechnology, Santa Cruz, CA), acetylhistone H3, acetyl histone H4 (EMD Millipore, Billerica, MA), alpha smooth muscle actin (αSMA Dako, Carpinteria, CA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology), followed by alkaline phosphatase conjugated anti-mouse, anti-goat, or anti-rabbit secondary antibody. After washing three times in 0.05% Tween-20 in Tris-buffered saline of pH 8.0 for 5 min each, the blot was developed using the nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate method. Three separate western blots were performed for each experiment. The digital quantification of western blots was performed using NIH Image J software.