2.6. BRAF Kinase Assay

The activities of silybins and dehydrosilybins were tested using a commercial BRAF (V600E) (serine/threonine-protein kinase B-raf, V600E mutant) Kinase Assay Kit (#48688, BPS Bioscience, San Diego, CA, USA). Briefly, the BRAF (V600E) enzyme was incubated for 45 min at 30 °C with five different concentrations of each tested compound in logarithmic dilutions (1000, 100, 10, 1, and 0.1 µM). The reaction mixture was prepared following the instructions of the producer. The luminescence of the product of the enzyme activity was measured with the Kinase-Glo MAX Kit (#V6071, Promega, Madison, WI, USA) at a microplate luminometer (Synergy Multi-Mode Reader, BioTek, Winooski, VT, USA). Higher luminescent signal corresponded to higher enzyme activity. Vemurafenib (PLX4032, #S1267, Selleck Chemicals, Houston, TX, USA) applied in concentrations 0.1, 1, 10, 100, and 1000 nM served as a reference compound. The reaction mixture without the test inhibitor represented the positive control, whereas the mixture without the test inhibitor and without the enzyme was used as blank.