Anti smad3

Cells were fixed for 10 min at 4°C in 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 10 min at room temperature, and blocked for 1 hour in PBS with 1% horse serum (Sigma-Aldrich, St. Louis, MO). The primary antibodies used were as follows: anti-NANOG (1:400; cat. no. 4903, Cell Signaling), anti-SOX17 (1:100; cat. no. 81778, Cell Signaling), anti-FOXA2 (1:100; cat. no. 685802, BioLegend), anti-SMAD2 (1:100; cat. no. 3122, Cell Signaling), anti-SMAD3 (1:100; cat. no. 9523, Cell Signaling), anti–p-SMAD2 (1:100; cat. no. 3108, Cell Signaling), and anti–p-SMAD3 (1:100; cat. no. 9520, Cell Signaling). The treated cells were subjected to three washes with PBS and further incubation with Alexa Fluor secondary antibodies (1:500; Jackson ImmunoResearch) for 1 hour at room temperature in the dark. The cells were then washed three times with PBS, with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO) added to the first wash to stain the nuclei. Images were acquired using a Confocal Zeiss LSM880. Intensity analysis was performed using ImageJ 1.51j8 (National Institutes of Health, MD, USA). For time-lapse imaging, cells were placed on an inverted microscope (Nikon, Ti-U inverted microscope system) and recorded every 2 hours for 72 hours in an environmental chamber maintained at 37°C with 5% CO₂.

The antibodies used in the present study included anti-human E-cadherin (1:500, Santa Cruz Biotechnology Inc.), anti-human N-cadherin (1:1,000, BD Bioscience), anti-α-SMA (1:300, Abcam, Cambridge, UK), anti–β-actin (1:5,000, Sigma-Aldrich), anti-Smad3, anti-pSmad3 (1:1,000, Cell Signaling Technology, Danvers, MA, USA), anti-ERK1/2, anti-pERK1/2 (1:1,000, Cell Signaling Technology), anti-p38 mitogen-activated protein kinase (MAPK), anti-pp38 MAPK (1:1,000, Cell Signaling Technology), anti-TβRI (1:1,000, Santa Cruz Biotechnology Inc.), and anti-TβRII (1:500, Abcam).

For this study, we used antibodies to PML (1:250 for IB, 1:100 for IP, sc-966, Santa Cruz Biotechnology, Delaware Ave., Santa Cruz, CA, USA), PML (1:500 for immunohistochemistry (IHC), sc5621, Santa Cruz Biotechnology, Delaware Ave.), HA (1:1000 for IB, 1:200 for IF, A190-108 A, Bethyl Laboratories Inc., Montgomery, TX, USA), β-actin (1:5000 for IB, A5441, Sigma-Aldrich, St Louis, MO, USA), Exportin-1 (CRM1; 1:5000 for IB, 1:2000 for IHC, A300-469A, Bethyl Laboratories Inc.), E-Cadherin (1:500 for IF, 610181, BD Transduction Laboratories, San Jose, CA, USA), EMT antibody Sampler Kit (9782, Cell Signaling Technology, Danvers, MA, USA), which contains rabbit antibodies each used at 1:300 (IB), N-Cadherin and Vimentin antibodies were used at 1:500 for IF, Phospho-Smad2/Smad3 (1:300 for IB, 8828, Cell Signaling Technology), Phospho-Smad2/3 (1:100 for IHC, sc-11769, Santa Cruz Biotechnology), Anti-rabbit IgG, HRP-linked Antibody (1:1000 for IB, 7074, Cell Signaling Technology), Anti-TGFβ Receptor I (1:1000 for IB, 3712, Cell Signaling Technology), Anti-SMAD2 (1:1000 for IB, 3122, Cell Signaling Technology), Anti-SMAD3 (1:1000 for IB, 9513, Cell Signaling Technology), Anti-phospho-SMAD2 (1:500, SAB4504207, Sigma-Aldrich), Anti-phospho-SMAD3 (1:500, SAB4300253, Sigma-Aldrich), Anti-mouse IgG, HRP-linked antibody (1:1000 IB, 7076, Cell Signaling Technology).