Ecl plus chemiluminescent detection reagent

HMCs with different treatment conditions were harvested and dissolved in protein extraction buffer containing protease inhibitor cocktails. The total protein from HMCs (80 µg) in all groups was subjected to SDS-PAGE on a 10% gel and then transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4 °C with mouse TGF-β1 (1:1000), Smad3 (1:1000), Smad4 (1:500), Smad7 (1:800), Collagen 1(1:1000), Rabbit p-Smad2/3(1:1000), anti-β actin (1:1000), or GAPDH (1:2000) antibodies in PBS before incubation with the appropriate peroxidase-labeled secondary antibodies. The reaction was detected with ECL plus chemiluminescent detection reagent (Amersham Pharmacia Biotech, Uppsala, Sweden). The images were scanned, and bands were quantified by Quantity One Software v4.62 (Bio-Rad, Hercules, CA, USA).

CoIP was performed from nuclear protein extracts. Nuclear protein from NIH-3T3 cells was prepared using the Ne-PER kit (Pierce) according to manufacturer's instructions. For each IP, 500–7500 μg nuclear protein was loaded in IP dilution buffer (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.5% NP40, 0.5 mM EDTA supplemented with protease inhibitors) and immunoprecipitated at 4C overnight with EZview Red anti-Flag M2 Affinity Gel (Sigma) according to manufactures instructions. Immune complexes were washed extensively and eluted first with 3X Flag peptide (Sigma) then in SDS sample buffer and boiled. Immunoprecipitated and input proteins were run on SDS-PAGE and immunoblotted with the following antibodies as indicated: goat polyclonal anti-KLF15 (Abcam) and anti-Flag (Sigma, Clone M2). Secondary HRP-conjugated antibodies and ECL-plus chemiluminescent detection reagent were from Amersham. Immunoblots presented are from a representative experiment that has been repeated three times.