HRECs were lysed in RIPA extraction buffer supplemented with protease and phosphatase inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA) [83 (link)]. Denatured samples (20 μg) were subjected to SDS-PAGE. Non-specific binding sites were blocked at room temperature for 1 h with 5% (w/v) Blotting-Grade Blocker (Bio-Rad Laboratories, Hercules, CA, USA) in Tris-buffered saline (Boston BioProducts, Boston, MA, USA) containing 0.05% (v/v) Tween-20 (Thermo Fisher, Waltham, MA, USA). Membranes were incubated overnight with the primary antibodies, anti-APEX1 (1:1000) (Novus Biologicals, Centennial, CO, USA; 13B8E5C2), and anti-vinculin (1:1000) (Novus; CP74-100), and then with the peroxidase-conjugated secondary antibody (1:1000) (Bio-Rad, 1706516) for 1 h. Signals were then captured by using a Bio-Rad ChemiDoc imager, and band intensities were analyzed by densitometry using Image Lab software (Bio-Rad).
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