Anti twist2

Western blots were performed as previously described [11] (link). Briefly, as soon as the harvested cells were lysed and the supernatant was collected, 60 µg protein was loaded onto SDS-PAGE and transferred to polyvinylidene fluoride membranes (PVDF). Membranes were blocked with 5% skimmed milk for 1 hour and incubated overnight with one of the following primary antibodies: anti-PSMD10 (diluted at 1∶1,000; Sigma, St. Louis, MO, USA), anti-Twist2 (diluted at 1∶200; Abcam, Cambridge, UK), anti-Vimentin (diluted at 1∶500; Cell Signaling Technology, Beverley, MA, USA), anti-β-catenin (diluted at 1∶500; Cell Signaling Technology), anti-E-cadherin (diluted at 1∶500; Cell Signaling Technology) and anti-cyclin D1(diluted at 1∶500; Cell Signaling Technology) rabbit monoclonal antibody, followed by 1 hour of incubation with the appropriate secondary antibody (1∶5,000). The anti-GAPDH (Epitomics) rabbit monoclonal antibody was diluted to 1∶1,000 for use as a sample loading control.

Serial sections (4 μm) subjected to immunohistological staining were fixed with freshly prepared 3 % H₂O₂ with 0.1 % sodium azide to quench endogenous peroxidase and then treated with antigen retrieval solution for 15 min. After placing in blocking reagent for 15 min, the sections were incubated in primary anti-HFI-2α (1:500, Abcam), anti-E-cadherin (1:1000, Abcam), anti-Twist1 (1:500, Abcam) and anti-Twist2 (1:350, Abcam) monoclonal antibody overnight at 4 °C, followed by incubation with the secondary antibody and Extravidin-conjugated horseradish peroxidase. The staining intensity was scored as: 0 (<10 %), 1 (10–50 %), 2 (>50 %). The final score was calculated by multiplication of the intensity score the quantity score. A score ≥ 2 was considered to represent positive expression.

Cell lysates were prepared as described previously^{22 (link)}. A total of 50 μg of protein was separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin for 1 h before being incubated overnight at 4 °C with the primary antibody, including anti-E-cadherin (Epitomics Biotechnology, 1:1000), anti-N-cadherin (Epitomics Biotechnology, 1:500), anti-Vimentin (Epitomics Biotechnology, 1:400), anti-TWIST2 (Abcam, 1:400), and anti-THBS2 (Novus Biotechnology, 1:800). β-actin (Abcam, 1:1000) was used as an internal control for protein loading and analysis. The membranes were washed three times with PBST (PBS buffer containing 0.1% Tween-20) and then incubated with HRP-conjugated secondary antibodies for 1 h. After the final wash with PBST, the antibody staining intensities were detected using ECL chemiluminescence reagents (Pierce).