2.4. Western blot analysis

Western blot analysis was carried out to measure the protein expression of CX43 in each sample. In brief, total protein was isolated from cell and tissue samples by making use of a radioimmunoprecipitation (RIPA) buffer (Thermo Fisher Scientific) following the standard experimental protocol provided on the operation manual by the buffer manufacturer. Then, the concentration of protein in each sample was determined by utilizing a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific) following the standard experimental protocol provided on the operation manual by the assay kit manufacturer. In the next step, the proteins were resolved by using 10% sodium dodecyl sulphate‐PAGE and then transferred onto a polyvinylidene difluoride membrane via electroblotting. In the next step, non‐specific binding was blocked with Tris‐buffered saline with Tween‐20 + 5% non‐fat milk before the membrane was probed overnight at 4°C with primary anti‐CX43 antibody (dilution 1:1000, OriGene Technologies) following the standard incubation protocol provided on the operation manual by the antibody manufacturer, followed by subsequent incubation with HRP‐conjugated secondary antibodies (dilution 1:5000, Thermo Fisher Scientific) for 1 hour at ambient temperature. After image development by making use of an enriched chemiluminescence assay kit (Thermo Fisher Scientific) following the standard experimental protocol provided on the operation manual by the assay kit manufacturer, the protein expression of CX43 in each sample was calculated.