4.5.1. Western Blots

Western blot was performed according to standard protocols as in [56] Mirzaii-Dizgah et al. (2020) with some modifications. The total protein was quantified using a bicinchoninic acid assay kit (Bioquochem, Oviedo, Spain), and an equal amount of protein was separated using a sodium dodecyl sulphate-polyacrylamide gel and then the proteins were transferred to a nitrocellulose membrane (Thermo Fisher Scientific, Waltham, MA, USA). The membrane was then blocked for 1 h at room temperature using 3% bovine serum albumin (BSA) before incubating it overnight with either ICAM-1 or SIRT1 primary antibodies (Abcam, Cambridge, UK). The membrane was washed three times with washing buffer (Tween-20/Tris-buffered saline) before incubating it with the respective secondary antibody (Mybiosource, San Diego, CA, USA) for 1 h at room temperature. Then, the membrane was washed three times before incubating it with ECL substrate (ThermoScientific, USA) for one minute and imaging it using the chemiLITE Chemiluminescence Imaging System (Cleaver Scientific, Rugby, UK). Equal gel loading was determined using actin as a housekeeping gene (Mybiosource, USA). The intensity of the bands was measured using Image J software (National Institutes of Health, USA, https://imagej.net/ij/docs/index.html, accessed on 12 September 2023).