2.6. PKC Kinase Activity Assay Kit

A PKC Kinase Activity Assay Kit (ab139437) was used for PKC detection. The manufacturer’s instructions were followed. First, 50 µL of Kinase Activity Assay buffer was added to each well at room temperature for 10 min after aspirating the liquid from all wells. Then, 30 µL of the samples were added, 10 µL of reconstituted ATP was added to each well and it was incubated at 30 °C for up to 30 min. The reaction was stopped by emptying the contents of each well. Subsequently, 40 µL PKC phosphorspecific substrate antibody was added to each well at room temperature for 60 min, and wash buffer was added to each well three times. Then, 40 µL of the diluted anti-rabbit IgG-HRP conjugate was added, it was incubated at room temperature for 30 min, and wash buffer was added to each well three times. Finally, 60 µL TMB substrate was added to each well, and 20 µL stop solution was added to each well. The absorbance increase was measured on a microplate reader at OD = 450 nm.