Hisol ecl plus detection kit

Endothelial cells were lysed in CETi lysis buffer (TransLab, Daejeon, Korea) on ice for 30 min and centrifuged at 13,000 rpm for 15 min. Supernatants were collected, and protein concentrations were determined at 595 nm using a protein assay kit (Pro-Measure, iNtRON Biotechnology, Seongnam, Gyeonggi, Korea). Equal amounts of total cellular proteins were boiled for 5 min and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto nitrocellulose membranes and incubated with primary antibodies followed by incubation with anti-mouse or anti-rabbit secondary antibody as appropriate. Finally, the densities of protein bands were measured using an enhanced Hisol ECL Plus Detection Kit (BioFact, Daejeon, Korea). Antibodies against p-eNOS, p-CaMKII, p-AMPK, and p-CaMKKβ, as well as anti-mouse and anti-rabbit IgG antibodies, were purchased from Cell Signaling Technology (Beverly, MA, USA). An antibody against β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

RAW264.7 cells were harvested and lysed with CETi Lysis Buffer (TransLab, Daejeon, Korea) for 30 min to extract total protein and centrifuged at 13,000 rpm for 15 min. Nuclear and cytoplasmic proteins were isolated using nuclear extract kits (Active Motif, Carlsbad, CA, USA). The supernatant was collected, and the protein concentration was determined at 595 nm using a Pro-Measure protein assay kit (Intron Biotechnology, Seongnam, Korea). Equal amounts of total cellular proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. After blocking with 5% skim milk, blots were incubated with the primary antibodies overnight at 4 °C followed by the corresponding secondary antibodies. Protein bands were visualized using a Hisol ECL Plus Detection Kit (BioFact, Daejeon, Korea). ImageJ software (NIH, Bethesda, MD, USA) was used to calculate the integrated optical densities of protein bands, which were normalized to that of the internal control.