Total cellular and corneal protein was extracted using a Minute Total Protein Extraction Kit (Invent Biotechnologies). A bicinchoninic acid protein assay kit (Millipore) was used to measure the total protein concentration. Equal amounts of protein samples were then loaded onto sodium dodecyl sulfate-polyacrylamide gels and electrophoresed. The separated proteins in the gels were transferred to polyvinylidene fluoride membranes (Millipore). After blocking with 5% nonfat milk in Tris-buffered saline with Tween 20 for 2 hours at room temperature, the membranes were incubated overnight with appropriate primary antibodies (Piezo1, 15939-1-AP; Proteintech, Wuhan, China; Pyrin [phospho S241], ab200420, Abcam, Cambridge, UK; Pyrin, ab195975, Abcam, Cambridge, UK; Phospho-RhoA [Ser188], AF8020-100, Affbiotech, Jiangsu, China; RhoA, 10749-1-AP, Proteintech, Wuhan, China; and β-actin, ab130935, Abcam, Cambridge, UK). After thorough rinsing, secondary antibodies (Abcam) were added and incubated for 1 hour at room temperature. Horseradish peroxidase signals were amplified using an ECL kit (Vazyme) and detected using a Bio-Rad Western blot detection system (Bio-Rad Laboratories, Inc.). Grayscale images of the Western blots were used for semiquantitative analysis with ImageJ software.