Recombinant human tgf β1

HK2 cells from Bmi-1-knockdown and negative control groups were seeded at 70% confluence in 1640 medium supplemented with 5% fetal bovine serum (FBS), 100 U ml⁻¹ penicillin, 100 µg ml⁻¹ streptomycin and 2 mM L-glutamine. Twenty-four hours later, the cells were changed to serum-free medium with 4 ng ml⁻¹ recombinant human TGF-β1 (Novoprotein Scientific Inc., Shanghai, China) for 24 h, then changed to 1640 medium supplemented with 5% fetal bovine serum (FBS), 100 U ml⁻¹ penicillin, 100 µg ml⁻¹ streptomycin and 2 mM L-glutamine for 60 h^{42 (link)}.

A human retinal pigment epithelial cell line (ARPE‐19) was purchased from American Type Culture Collection (ATCC, Manassas, VA, US) and cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F‐12 (DMEM/F‐12; Gibco, Grand Island, NY, US) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, US) at 37°C in a humid atmosphere containing 5% CO2. Recombinant human TGFβ1 was purchased from Novoprotein. SKL2001, the agonist of wnt/β‐catenin pathway, was purchased from MedChemExpress.
The pCDH‐Vec control vectors and pCDH‐METTL3 overexpression vectors as well as pLKO.1‐TRC‐Vec control vectors and pLKO.1‐TRC‐shMETTL3 knockdown vectors used for stable transfection were kindly provided by Prof. Shuibin Lin.¹⁸ Plasmids were transfected into ARPE‐19 cells using lentivirus, as described previously. Stably transformed ARPE‐19 cells were selected by selection on media containing 2  μg/ml puromycin (Solarbio Life Science, CN) for 48 h.²⁸

Antibodies to proteins were obtained from the following sources: EphB1 (#ab129103) and phos-EphB1 (#ab61791) for Western blot were purchased from Abcam; EphB1 (#AF542) for immunohistochemistry was purchased from Novus Biologicals; N-cadherin (#13116) and E-cadherin (#3195) were from Cell Signaling Technology; β-actin (#60008-1-Ig), Smad2 (#12570-1-AP) and GAPDH (#60004-1-Ig) were from Proteintech. Recombinant human Ephrin-B2 Fc chimera protein was purchased from R&D (7397-EB, RD Inc, MN, USA) and Recombinant Human TGF-β1 (#CA59) was purchased from Novoprotein.