Western Blot Analysis of Protein Expression

For Western blot analyses, proteins were isolated 72 h after transfection as previously described [62 (link)]. For electrophoresis, a 4% to 20% precast gradient gel (Bio-Rad Laboratories, Feldkirchen, Germany) was loaded with 30 µg of whole protein extract and 15 µg of a prestained protein standard (ThermoFisher Scientific, Waltham, MA, USA) for molecular weight evaluation. Electrophoresis, protein transfer, and antibody staining were performed as previously described [10 (link)]. Incubation with the primary antibody was performed overnight at 4 °C and with the secondary antibody for two hours at room temperature. Antibody details are specified in Supplementary Table S4. Chemiluminescence of bound antibodies was induced using ECL substrate (ThermoFisher Scientific, Waltham, MA, USA) and visualized on X-ray films. Afterwards, the membrane was stripped and then stained again to visualize another protein of interest. The intensity of specific bands was measured with ImageJ Ver. 1.53 (NIH, Bethesda, MD, USA) and normalized to tubulin as the reference protein. Importantly, samples and respective controls were stained on the same gel.