Anti p smad2

Western blot analysis was performed according to a standard protocol.⁴⁴ (link) The primary antibodies included anti-ACP5 (Gentex, CA, USA, 1:1,000), anti-fibronectin, anti-E-cadherin, anti-vimentin (Proteintech, Wuhan, China, 1:1,000), anti-p38, anti-ERK, anti-p-ERK, anti-AKT, anti-β-catenin, anti-SMAD2/3, anti-p-SMAD2, anti-p-SMAD3, anti-p53, anti-p53 (Ser392), anti-ubiquitin (Cell Signaling Technology, Danvers, MA, USA, 1:1,000), anti-GAPDH, and anti-β-actin (Abcam, Cambridge, MA, USA, 1:3,000) antibodies. Detection was performed using a chemiluminescent substrate system (Bio-Rad, Hercules, CA, USA). The gray values were analyzed with ImageJ software.

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.

Whole cell extracts were prepared using RIPA buffer, resolved on SDS-PAGE gels, and transferred to acetate cellulose membranes. Primary antibodies used were anti-TAGLN (1:1000, Abcam plc.), anti-MYH11 (1:500, Abcam plc.), anti-ACTA2 (1:200, Sigma Corp.), anti-CNN1 (1:500, Sigma Corp.), anti-pSMAD2 (1:500, Cell Signaling Technology Inc.), anti-pS6 (1:500, Cell Signaling Technology Inc.), anti-GAPDH (1:2000, Santa Cruz Inc.). Secondary antibodies used were IRDye680RD Donkey anti-Mouse, IRDye680LT Donkey anti-Rabbit, IRDye800CW Donkey anti-Mouse, IRDye800CW Donkey anti-Rabbit (All secondary antibodies were from Licor Inc.). Licor western blot detection system was used for the dual-color imaging. ImageJ was used for the quantification of bands.
For the ELISA assay of the secreted TGF-β1, fresh medium (DMEM/F12, N2, Pen/Strep, all from Life technologies Corp.) without serum was applied to the cells and harvested after 24 h. ELISA assay was carried out using the Human TGF-beta 1 Quantikine ELISA Kit from R&D systems following the manufacture's guidelines.