Western blot analysis of SDPR and CAV1 expression in healthy TM tissues

To evaluate SDPR and CAV1 protein expression of healthy TM tissues, corneal tissues were collected from both B and W donors. Total TM strips were dissected into smaller fragments and lysed using 60 μl of RIPA buffer. Subsequently, approximately 12 μg of protein per sample/lane was separated using electrophoresis on 4–12% gradient gels. The separated proteins were then transferred onto nitrocellulose membranes using the Mini Gel Tank (Cat. No. A26977, Thermo Fisher Scientific) and Mini Blot Module (Cat. No. B1000, Thermo Fisher Scientific).

For immunodetection, primary antibodies SDPR (rabbit polyclonal antibody, Thermo Fisher Scientific) at a dilution of 1:1000 and CAV1 (mouse monoclonal antibody, Thermo Fisher Scientific) at 1:200 was added to the membranes and incubated overnight at 4 °C. Subsequently, secondary antibodies IRDye® 800CW Donkey anti-Rabbit IgG and anti-Mouse (LI-COR, Nebraska) were used at a dilution of 1:20,000. The Western blot signals were visualized using the Odyssey Near-Infrared Western Blots System (LI-COR, Nebraska). To ensure accuracy and consistency, intensity of the bands was normalized to the expression level of β-actin on the same blots, using a mouse monoclonal antibody (1:10,000; Santa Cruz). The averages of blot band intensity were quantified using LI-COR software Image Studio. Normalization was performed by adjusting the intensity of the SDPR and CAV1 bands based on the level of β-actin values, and then setting the highest normalized intensity for SDPR/CAV1 to 1.0 for each group. To obtain reliable results, duplicate blots were used for each sample. The data were normalized, averaged, and presented ± standard deviation (SD).