Western blot analysis of signaling pathway protein expression

MDA-MB-231 cells were treated with 5 ng/ml TGF-β1 and total protein was extracted with cell lysis buffer (Thermo Fisher Scientific, Inc.), 25 µg protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the proteins were transferred to film. The polyvinylidene difluoride membrane was blocked by non-fat milk at 37°C for 24 h, and then incubated overnight at 4°C with the following antibodies: Rabbit polyclonal anti-Smad2 (dilution, 1:1,000; catalog no., ab192175; Abcam, Cambridge, UK), rabbit polyclonal anti-p38 (dilution, 1:1,000; catalog no., ab38238; Abcam), rabbit polyclonal anti p-Smad2 (dilution, 1:800; catalog no., 3101; Cell Signaling Technology, Inc., Danvers, MA, USA) and rabbit monoclonal anti p-p38 (dilution, 1:800; catalog no., 9215; Cell Signaling Technology, Inc.). The membrane was then incubated with horseradish peroxidase-conjugated goat anti-mouse polyclonal secondary antibody (dilution, 1:5,000; catalog no., ab6789; Abcam) or horseradish peroxidase-conjugated goat anti-rabbit polyclonal secondary antibody (dilution, 1:5,000; catalog no., ab6721; Abcam) at room temperature for 2 h. The protein bands were visualized using DAB reagent (BD Biosciences), and images were captured using a Vilber-Fusion chemiluminescence system (Molecular Imaging Vilber Fusion X7; Vilber Lourmat Deutschland, Eberhardzell, Germany) and analyzed using Image J software version 1.48 (imagej.nih.gov/ij/index.html). The optical density ratio comparing the expression of each target protein prior to and following TGF-β1 treatment was recorded, and β-actin was used as a control. The experiment was repeated 3 times.