Western Blot Analysis of Cav1.3 Protein

Samples were collected and prepared as described previously (Ko M. L. et al., 2009 (link); Huang et al., 2013 (link); Lin et al., 2015 (link)). Retinas were homogenized in a Tris lysis solution (50 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% NP-40) including phosphatase (50 mM NaF, 1 mM Na₃VO₄) and protease inhibitors (Sigma-Aldrich). After centrifugation to remove cellular debris, an equal volume of 2x Laemmli buffer was added to each sample lysate, then the samples were heated at 95°C for 5 min. Proteins were separated by SDS-PAGE (10% gels) for 1–2 h. Proteins were then transferred to nitrocellulose membranes and probed by primary antibodies. The primary antibodies used were rabbit anti-Ca_v1.3 antibody (1:1,000; Chemicon/ Millipore Sigma) and rabbit anti-actin antibody (1:1,000; Cell Signaling Technology, Danvers, MA, USA). Actin was used for loading controls. The secondary antibody (goat anti-rabbit) conjugated to horseradish peroxidase (1:1,000; Cell Signaling Technology) and the Femto and Pico electrochemiluminescense (ECL) kits (Pierce ThermoFisher Scientific, Waltham, MA, USA) were used to visualize the blots.