Anti phospho smad2

Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA), emodin was obtained from Shanghai future industry Limited by Share Ltd (Shanghai, China) and BLM was acquired from Nippon Kayaku (Tokyo, Japan). The primary antibodies described in the study include: anti-E-cadherin, anti-vimentin, anti-cleaved caspase-3, anti-phospho-Smad2, anti-phospho-Smad3, anti-Smad2, anti-Smad3, anti-phospho-Erk1/2 and anti-Erk1/2 (Cell Signaling Technology, CA, USA); anti-caspase-3, anti-Bax (Santa Cruz Biotechnology, CA, USA); anti-fibronectin (Proteintech, Chicago, USA); anti-caspase-8, anti-Bcl-2 (Absci, MD, USA); anti-TGF-β1, anti-FSP-1, anti-α-SMA (Abcam, USA); and anti-GAPDH (Beyotime Institute of Biotechnology, Haimen, China). Other reagents were obtained from Beyotime Institute of Biotechnology unless otherwise indicated.

Oral tissues were fixed by neutral-buffered formalin and embedded in paraffin to assess SMAD2 phosphorylation. After preparation of thin sections, slides were deparaffinised with xylene, followed by rehydration through graded alcohol to water. Antigen retrieval was performed in target retrieval solution (pH 9.0) at 120 °C for 15–20 min (S2367;  Dako). Then, endogenous peroxidase was quenched with 3% (v/v) H₂O₂ for 5 min. After blocking with 1% bovine serum albumin (BSA) at room temperature for 2 h, the sections were incubated with a primary antibody diluted with 1% BSA in phosphate-buffered saline (PBS). Slides were incubated overnight at 4 °C with primary monoclonal antibodies. Primary antibody was as follows: anti-phospho-SMAD2 (1:100, 3108; Cell signalling Technology). Slides were then incubated with horse radish peroxidase using a ChemMate EnVision kit (Dako) for 2 h and washed twice with PBS. Immunoreactivity was visualised with 0.6 nm 3,3′-diaminobenzidine tetrahydrochloride (Dojindo) and counterstained with haematoxylin. Images were acquired with a BX53 microscope and DP72 microscope digital camera (Olympus) and analysed using Olympus cellSens software. ImageJ software was used to quantify phosphorylated-SMAD2 (p-SMAD2) protein levels.