Smad2 3 sc133098

Human colorectal cancer-derived cell line HCT116 cells were transduced with Lv.TβRII-SE/Fc at MOI 200, in the presence of 8 μg/ml polybrene (Millipore Sigma, Burlington, MA, United States). HCT116 cells (1 × 10⁶) overexpressing TβRII-SE/Fc or control were seeded in 60-mm cell culture dishes and starved for 24 h in DMEM. After that, cells were incubated in DMEM ± 5 ng/ml TGF-β1 for 1 h. Cells were lysed in RIPA buffer (Millipore Sigma, Burlington, MA, United States) supplemented with 1 mM PMSF and quantified by Bradford Assay. Proteins were separated by electrophoresis on 10% SDS-PAGE gels and electrotransferred onto Immobilon-polyvinylidene difluoride membranes (Millipore Sigma, Burlington, MA, United States). After blocking with 5% non-fat milk, membranes were probed with antibodies for P-Smad2/3 (sc-11769) and Smad2/3 (sc133098) (Santa Cruz Biotechnology, Inc., Dallas, TX, United States) at 4°C overnight and then incubated with horseradish peroxidase (HRP)-conjugated secondary anti-mouse or rabbit antibodies (Thermo Fisher Scientific, Waltham, MA, United States). Protein expression was detected by using an enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific, Waltham, MA, United States). Densitometry was performed using ImageJ software (National Institutes of Health, Bethesda, MD, United States).

We used 5 µm eye sections for the immunohistochemistry after paraffin imbibition, deparaffinization, and rehydration. The primary antibodies used were rabbit polyclonal TGF-β1 (sc-146; Santa Cruz Biotechnology; Dallas, TX, USA) and Smad2/3 (sc-133098; Santa Cruz Biotechnology; Dallas, TX, USA). These were diluted to 1:200 and incubated overnight at 4 °C. Immunoreactions were detected using a Novocastra Peroxidase/DAB kit (Leica Biosystems, Nussloch, Germany). The negative control sections were stained with irrelevant immunoglobulins and analyzed under a bright-field microscope [37 (link)]. The percentage of positive-stained area/total area was quantified using ImageJ software 1.47.

Dulbecco’s modified Eagle medium (DMEM), minimum essential medium-alpha (α-MEM), Dulbecco’s modified Eagle medium:nutrient mixture F-12 (DMEM/F-12) without phenol red, Dulbecco’s phosphate buffered saline (PBS), fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS), antibiotic-antimycotic mixture containing 100 U/mL penicillin and 100 U/ mL streptomycin, Opti-MEM, Geneticin (G418), and 0.25% trypsin-EDTA were purchased from Gibco BRL (Grand Island, NY, USA). Recombinant human chemerin (Glu21-Ser157, with an N-terminal Met), mouse soluble receptor activator of nuclear factor kappa beta (RANK) ligand (RANKL), and macrophage colony-stimulating factor (M-CSF) were purchased from R&D Systems (Minneapolis, MN, USA). Anti-human E-cadherin (sc-8426), β-catenin (sc-1496-R), vimentin (sc-6260), lamin B (sc-6216), RANKL (sc-377079), osteoprotegerin (OPG; sc-71747), Smad2/3 (sc-133098), and GAPDH (sc-32233) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Transforming growth factor (TGF)-β and insulin-like growth factor (IGF)-1 were purchased from Peprotech (Rocky Hill, NJ, USA). Histopaque-1083, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents used in this study were of analytical grade.