Rabbit monoclonal anti smad2 3

Cells were lysed in 50 mM Tris, 150 mM NaCl, 5 mM EGTA and 0.75% NP40 (pH 7.5), supplemented with Complete Protease Inhibitor Cocktail, 2 mM Na₃VO₄, 50 mM NaF, and 10 mM NaPPi. Cells debris was pelleted and protein concentration in the supernatant was determined using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL,USA). Proteins were separated by SDS−PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the following primary antibodies: mouse monoclonal phospho-p38 (Thr180/Tyr182) antibodies, rabbit polyclonal p38 antibodies, rabbit monoclonal anti-phospho-Smad2 (Ser 465/467)/ Smad3(Ser 423/425), rabbit monoclonal anti Smad2/3, (all purchased from Cell Signaling, Danvers MA, USA), and monoclonal mouse GAPDH antibodies (Millipore, Billerica MA, USA). Following incubation with the appropriate secondary peroxidase-conjugated antibodies (Jackson, West Grove, PA, USA), immunopositive bands were visualized by an enhanced chemiluminescence (ECL) detection kit (Pierce, Rockford, IL, USA) using the Molecular Imager ChemiDoc XRS+ System (Bio-Rad, Hercules, CA, USA). The optical density of each protein band was quantified by the Image-Lab analysis software (Bio-Rad, Hercules, CA, USA). The amount of GAPDH in each lane was determined as a loading control.

HLMFs were grown in T75 flasks, serum-starved for 24 h, and stimulated with TGF-β1 (10 ng/ml) in the presence of either serum-free medium alone, 0.1% ethanol control, LXA₄ 10⁻¹⁰ mol, or LXA₄ 10⁻⁸ mol for 1 h. HLMFs were detached with 0.1% trypsin/EDTA and washed. Protein was isolated using the RIPA buffer lysis system (Santa Cruz Biotechnology), and total protein concentration was determined using the DC Bio-Rad protein Assay (Bio-Rad). A total of 30 μg protein was resolved using 10% Mini-Protean TGX precast gels (Bio-Rad) and then transferred to an Immunobilon-P polyvinylidene difluoride membrane using Transblot Turbo transfer packs (Bio-Rad). Membranes were blocked with 5% milk and incubated with rabbit monoclonal anti-phosphorylated-Smad2/3 (0.231 μg/ml; Cell Signaling Technology) or rabbit monoclonal anti-Smad2/3 (0.0087 μg/ml; Cell Signaling Technology). Protein bands were identified by HRP-conjugated secondary Ab and ECL reagent (Amersham). Immunolabeled proteins were visualized using ImageQuant LAS 4000 (GE Healthcare Life Sciences).