Western Blot Assay

To determine the change in autophagy, we detected the expression of the autophagy-associated markers LC3II/I and P62 by Western blotting. Protein samples from mouse corneal stromal cells or corneal tissues were extracted using RIPA buffer reagent (Thermo Fisher Scientific, Germany) and separated using 12.5% SDS-PAGE gels. After electrophoresis, the separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% defatted milk powder (Applygen, Beijing, China). Primary antibodies, including anti-LC3 I/II (L7543; Sigma, St. Louis, MO, USA), anti-p62 (P0067; Sigma), anti-IL-1β (ab9722; Abcam, Cambridge, UK), anti-ATG5 (A0856; Sigma), and anti-GAPDH (AF7021; Affinity Biosciences, Cincinnati, OH, USA), were incubated overnight at 4°C. After washing with PBS containing 0.05% Tween 20 (Bio-Rad, Hercules, CA, USA) three times, the membranes were incubated with specific secondary antibodies for 2 hours. Finally, the proteins were quantified using the ECL detection reagent (WBKLS0100; Millipore, Billerica, MA, USA).