Western Blot Protein Analysis Protocol

Whole-cell lysates were obtained as previously described (Di Tinco et al., 2021 (link)). A measure of 30 µg of protein extract for each sample was quantified by Bradford protein assay (Bio-Rad), and separation was performed by SDS-polyacrylamide gel electrophoresis on Mini-PROTEAN^® TGX™ Stain-Free Precast gels. Gels were then UV-activated using a ChemiDoc MP Imaging System (Bio-Rad), and proteins were subsequently transferred to 0.2-μm nitrocellulose membranes (Bio-Rad). The membranes were then imaged using the ChemiDoc Imaging System (Bio-Rad) for total protein normalization. Membranes were incubated overnight with the following primary antibodies: mouse anti-α-SMA (Invitrogen), mouse anti-fibronectin (Invitrogen), rabbit anti-PPARγ (Cell Signaling Technology), rabbit anti-PDGFRβ (Cell Signaling Technology), and rabbit anti-GLI1 (Invitrogen), all diluted 1:1,000 in 0.1% TBS-Tween 20 (Sigma-Aldrich) and then were incubated with HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (1:3,000; Thermo Fisher Scientific) for 30 min at room temperature. The membranes were visualized using a ChemiDoc Imaging System (Bio-Rad). Finally, the relative expression levels of each evaluated marker were then obtained by normalizing the density of the protein bands to the corresponding stain-free blot image using Image Lab software (Version 6.1, Bio-Rad).