Anti vinculin

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).

For whole cell protein isolation, cells were harvested in RIPA buffer a containing protease inhibitor cocktail (Sigma) or phosphatase inhibitor cocktail (Sigma) for phosphorylated proteins. Followed by sonication and centrifugation, the protein concentration of the supernatant was determined by Lowry protein assay. The lysates were run on a 10% SDS-PAGE gel and transferred to a 0.45 μm nitrocellulose membrane for 4 h at 300 mA. After blocking in 5% milk in TBST buffer for 1 h at room temperature, the membranes were incubated with anti-TLR4 (Santa Cruz sc-293072), anti-p-IκB-α (Santa Cruz sc-8404), anti-IκB-α (Santa Cruz sc-1643), anti-Na⁺/K⁺-ATPase α1 (Santa Cruz sc-21712), anti-LAMP1 (Santa Cruz sc-20011) or anti-PMCA (plasma-membrane-type Ca2⁺-ATPases) (Santa Cruz sc-271917), along with anti-vinculin (Novus NB600-1293) antibodies or anti-β-Actin antibody (Santa Cruz sc-47778) as protein loading control.