Evaluating TGF-β1 Expression by Western Blot

A Western Blot (WB) investigation was carried out to evaluate TGF-β1 protein content in samples’ CM. In detail, equal volume amount (30 µL) of CM collected from CTR, and cells seeded on PLGA fleeces and 3D scaffolds, were processed for WB as previously described [38 (link)]. The total protein amount in CM has been normalized on a Ponceau S stain, according to Sander et al. [39 (link)]. TGF-β1 expression was detected using a specific primary antibody (Abcam, ab27969, Milan, Italy) diluted (1:250) in tris-buffered saline (TBS) solution, and incubated overnight at 4 °C. Finally, the WB membranes were incubated for 1 h at RT with an anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP) (Santa Cruz, sc-516102, Heidelberg, Germany) diluted in TBS (1:10,000). The ECL substrate was used to visualize the target protein (LiteAblot PLUS, Euroclone, Milan, Italy) and Azure’s 400 detected the chemiluminescent signal (Azure Biosystems, c400, Sierra Ct, Dublin, CA, USA). Image J blot analyzer software was employed for the densitometric analysis (ImageJ 1.53k, NIH, Bethesda, MD, USA).