Fig. 2.

Epithelial–mesenchymal transition increases rVAR2 binding. a Western blot of A549 cell lysates after 24, 48, and 72 h of treatment with TGF-β or control (TGF-β dissolution buffer). Blots were incubated with rabbit anti-E-cadherin, anti-vimentin, anti-N-cadherin, or anti-GAPDH antibodies and detected by anti-rabbit HRP antibody. b Representative images of fixed A549 cells after 48 h of treatment with TGF-β or control buffer. Cells were incubated with DAPI, phalloidin alexafluor 594 to stain F-actin, anti-pan Cytokeratin Alexa Fluor 488, or rabbit anti-E-cadherin and anti-vimentin in combination with anti-rabbit FITC antibodies. Scale bars, 50 µm. c Intensity of rVAR2 staining (MFI) of A549 cells treated with TGF-β or control buffer for 48 h (P < 0.001, generalized least squares regression model). Binding of rVAR2 was detected by flow cytometry using anti-penta His Alexa Fluor 488 antibody. Three independent experiments were performed. MFI mean fluorescence intensity