Horseradish peroxidase hrp conjugated secondary anti mouse or rabbit antibodies

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).