Anti p 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).

All transfected cells were seeded into 6-well tissue culture plates (1–2×10⁶) at 37°C and lysed using 200 µl protein (RIPA; Sangon Biotech Co., Ltd., Shanghai, China). The protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Sangon Biotech Co., Ltd.). The proteins were separated using 12% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.). Following blocking of the membranes using 5% nonfat milk in TBST, the membranes were incubated with anti-p-SMAD3 (#9520; dilution 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-TGF-β (#3709; dilution, 1:2,000; Cell Signaling Technology Inc.) and β-actin (#4970; dilution 1:2,000; Cell Signaling Technology, Inc.) antibodies at 4°C overnight. Following washing with TBST, the membranes were incubated with a goat anti-rabbit secondary antibody (#A16110; 1:5,000; Pierce; Thermo Fisher Scientific, Inc.) at 37°C for 1 h, and the blots were detected using an enhanced chemiluminescent reagent (#G-21234; Pierce; Thermo Fisher Scientific, Inc.) and quantified using Image Lab 3.0 (Bio-Rad Laboratories, Inc.).

Cells transfected with pCMVβ-myc3-MDM2 plasmid or empty vector were plated in 6-well plates. Then cells were fixed with 4% paraformaldehyde in PBS for 15 min, and blocked with 3% bovine serum albumin (Sigma-Aldrich, A7906) and 0.3% Triton X-100 in PBS for 1 h at room temperature. MDM2 and p-Smad3 was probed with primary antibodies anti-MDM2 (1 : 50) and anti-pSmad3 (1 : 150) in PBS overnight at 4 °C at room temperature and Alexa Fluor 488 donkey anti- rabbit IgG secondary antibody (Invitrogen, A-11008) or Alexa Fluor 568 donkey anti-mouse IgG secondary antibody (Invitrogen, A-10037) diluted in 1 : 500 in PBS for 1 h at 37 °C. Nuclei were visualised by staining with DAPI (4′ 6-diamidino-2-phenylindole; Sigma-Aldrich, D9542). Cells were than washed thrice with PBS and imaged with Leica DMI 400B fluorescence microscope.
anti-MDM2 (Cat# sc-965), anti-E-cadherin (Cat# sc-7870) were purchased from Santa Cruz (Santa Cruz), and anti-p-Smad3 (Cat# 9520) was from Cell Signaling Technology (Beverly, MA, USA).

Western blot was performed as previously described [19 (link)]. Membranes were incubated with anti-p-Smad3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-Smad3 (1:1,000; Cell Signaling Technology), anti-PAI-1 (1:1,000; BD Biosciences), anti-α-SMA (1:1,000; Sigma), and anti-type I collagen (1:1,000; Abcam) polyclonal antibodies at 4℃ with gentle shaking overnight. Antibodies were detected by horseradish peroxidase-linked secondary antibody (Santa Cruz) using an Enhanced Chemiluminescence Western Blotting Detection System, in accordance with the manufacturer's instructions (Millipore, Billerica, MA, USA) [19 (link)]. The membrane was reblotted with anti-β-tubulin antibody (Applied Biological Materials Inc., Richmond, BC, Canada) to verify equal protein loading in each lane. Densitometric measurements of the bands were performed using UN-SCAN-IT digitizing software (Silk Scientific Corp., Orem, UT, USA).