Connexin 26

Lysates were prepared with radioimmunoprecipitation assay (RIPA) buffer. Protein concentration in the samples was measured using bicinchoninic acid (BCA) protein assay kit and adjusted using water. Samples were loaded onto a 10% acrylamide gel. The gel was run at 120 V for 90 min. Afterward, membrane transfer was performed at 250 mA for 70 min. Membranes were incubated with primary antibodies against NDUFS1 (Thermo Fisher, Cat# PA5-22309), aldolase A (Novus Biologicals, Cat# NBP1-87488), NHE1 (BD Biosciences, Cat# 611775), β-actin (Proteintech, Cat# HRP-60008), and GAPDH (Proteintech, Cat# HRP-60004) used as a loading control; connexin 26 (Thermo Fisher, Cat# 13-5800), connexin 31 (Proteintech, Cat# 12880-1-AP), connexin 43 (Thermo Fisher, Cat# 13-8300), and HRP-conjugated goat anti-rabbit and anti-mouse secondary antibodies were applied. The membrane was visualized using ECL.

Proteins (30 μg) extracted from the cortices of five sham‐operated, six BDL, six HA, and five BDL‐OP treated rats were separated by way of sodium dodecyl sulphate‐polyacrylamide gel electrophoresis on a 4%‐12% Bis‐Tris NuPAGE gel (Invitrogen, Scotland, UK) and transferred to nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin and incubated with antibodies against connexin‐43 (Cell Signaling Technology, Danvers, MA; 1:1000), connexin‐36 (Santa Cruz Biotechnology, Dallas, TX; 1:1000), connexin‐30 (Invitrogen, 1 µg/mL), and connexin‐26 (Thermo Fisher Scientific, Waltham, MA; 1 µg/mL). Detection of actin (Santa Cruz Biotechnology; 1:1000) was used to control for protein loading. Binding of antibody was detected using a horseradish peroxidase–conjugated secondary antibody (goat anti‐rabbit or goat anti‐mouse IgG‐HPR, Santa Cruz Biotechnology; 1:10,000) where appropriate and the SuperSignal Chemiluminescence Substrate for detection of horseradish peroxidase (Pierce, Thermo Fisher Scientific, Waltham, MA). Densitometric analysis was performed using Kodak 1D image analysis software (Kodak, Rochester, NY).