Western Blot Analysis of Autophagy and Fibrosis Markers

Westrern blot assay was performed as previously described elsewhere [24 (link)]. Briefly, total proteins quantification was done through the BCA method (Beyotime, Shanghai, China). Subsequently, proteins (30 μg) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by a transfer into a membrane of polyvinylidene difluoride (PVDF) (Thermo Fisher Scientific). The membrane was later blocked for 1 hour using 5% skimmed milk in TBST, incubated using rabbit anti-human ATG5 (ab109490, 1:1000, Abcam), Beclin-1 (ab210498, 1:1000), LC3I/II (ab192890, 1:1000), p62 (ab109012, 1:1000), collagen I (ab138492, 1:1000), collagen III (ab184993, 1:1000), α-SMA (ab5831, 1:1000), at 4°C overnight. The membrane was subsequently incubated for 1 hr at room temperature using secondary antibody horseradish peroxidase-labeled goat anti-rabbit IgG (1:5000, Solabio). Finally, the band detection was done using the ECL Chemiluminescent Substrate Kit (Thermo Fisher Scientific). β-actin (4970S, 1:1000, Cell Signaling Technology) was as the loading control. Each experiment was performed in triplicate.