Fluozin 3 tetrapotassium salt

ZnSO4 (0 µM, 0.1 µM, 1 µM, 2 µM, 3 µM, 4 µM, 5 µM, 10 µM, 20 µM and 50 µM) and 1 µM Fluozin-3 tetrapotassium salt (ThermoFisher, Germany) were supplemented to HDAC1 buffer and 100 µL per well were distributed on a 96 well plate as triplicates. To determine minimal and maximal fluorescence as described before, 50 µM TPEN and 100 µM ZnSO4 were pipetted to the HDAC1 buffer as triplicates, as well (Fig. S3). Subsequently, samples were incubated for 30 min at 37°C in the dark, followed by fluorescence measurement at the fluorescence plate reader Ultra 384 (Tecan, Germany). To determine the maximal fluorescence within the PP2A buffer, 3 µM ZnSO4 were added. Due to the concentration optimum of FluoZin-3, the free zinc concentration for higher zinc concentrations was calculated with the respective formula afterwards. Free zinc concentrations in a higher range could not be reliably determined within the PP2A buffer. Fig. S3 A represents the free zinc in the reaction buffer with the experimental conditions wherein a 1/50 dilution of the PP2A preparation was used with a standard peptide as substrate whereas Fig. S3 B
shows free zinc concentrations in the reaction buffer wherein a 1/20 dilution of the PP2A preparation was tested with a more physiological Rb peptide as substrate.