Odyssey software version 5

Proteins were extracted from cell lysates in lysis buffer and used to quantify protein levels. Proteins were separated on polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoprobed with specific antibodies. The proteins were detected using a chemiluminescence reagent kit purchased from Thermo Fisher Scientific. The imager station captured the images (Odyssey Software Version 5.2, LI-COR Biosciences). Anti-TRIO (Affinity, DF2685), anti-TRIOBP (Proteintech, 16124-1-AP), anti-β-actin (Affinity, AF7018), and anti-GAPDH (Affinity, AF7021) antibodies were purchased from Affinity Biosciences LTD. Anti-α-SMA antibodies were purchased from Abcam (Abcam, ab124964). Anti-vimentin (Proteintech, 10366-1-AP), anti-E-cadherin (Cell Signaling Technology, 14472S), anti-type I collagen (Proteintech, 14695-1-AP) and anti-fibronectin (Proteintech, 15613-1-AP) antibodies were purchased from Proteintech Group.

Total proteins from conditioned medium or isolated from cell lysates were separated using polyacrylamide gel electrophoresis (12% acrylamide) under reducing conditions, as previously described (Kevenaar et al., 2008 (link); Hoyos et al., 2020 (link)) In brief, blots were incubated with the mouse monoclonal antibodies 5/6A (recognizing the C-terminal mature region) at a 1:1000 dilution (Serotec, Bio-Rad Laboratories BV, Veenendaal, The Netherlands), followed by the Alexa Fluor-800 goat anti-mouse antibody (Molecular Probes, Invitrogen, Breda, The Netherlands) at a 1:7500 dilution. The Licor-Odyssey imaging system was used for protein visualization and blots were analyzed using the Odyssey software version 5.2 (LI-COR Biosciences, Westburg, Leusden, The Netherlands).