Rabbit monoclonal anti smad2

Human tissue samples and cultured cells were lysed in radio-immunoprecipitation assay buffer (Beyotime, China) for protein extraction. In brief, 15–25 μg of the protein was first resolved by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis, and the bands were then electro-blotted onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% skim milk or 5% bovine serum albumin for 1 h and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-E-cadherin (1:2500), anti-vimentin (1:2000), anti-TGF-β1 (1:1000), rabbit monoclonal anti-SMAD2 (1:5000), rabbit monoclonal anti-phospho-SMAD2 (1:1000, Cell Signalling Technology, USA), and anti-GAPDH (1:20,000, Proteintech). Following this, the membranes were washed three times with phosphate-buffered saline Tween-20 (PBS-T) and incubated with HRP-conjugated AffiniPure goat anti-rabbit IgG (1:5000, Proteintech). Signals were visualised using the enhanced chemiluminescence reagent according to the manufacturer’s protocol.

Mouse monoclonal anti-β-ACTIN, rabbit polyclonal anti-phospho Smad2, rabbit monoclonal anti-Smad2, rabbit monoclonal anti-phospho Smad3, rabbit polyclonal anti-Smad3, rabbit polyclonal anti-TGF-β, rabbit monoclonal anti-PCNA, and rabbit monoclonal anti-SMURF2 were obtained from Cell Signaling Technology (Beverly, MA, USA). Rabbit polyclonal anti-CRIF1 was obtained from Abcam (Cambridge, UK). Rabbit polyclonal anti-SMAD7 antibody was obtained from Invitrogen (Carlsbad, CA, USA). For Western blot, 15 μg of whole cell lysates was loaded and separated on 6–12% SDS-PAGE gels by electrophoresis, followed by incubation in the appropriate primary and secondary antibodies. For each western blot quantified, the experiment was repeated for a minimum of three times. Blots were imaged using a chemiluminescence assay kit (Miracle-Star Western Blot Detection System; Intron Biotechnology, Seongnam, Republic of Korea) and EZ-Western Lumi Femto (Daeil Lab Service, Seoul, Republic of Korea), and band densities were quantified on a Gel Doc 2000 Chemi Doc system using Quantity One software (Bio-Rad, Hercules, CA, USA). Values were normalized to β-actin (loading control).