In vitro phosphorylation assay was performed as described elsewhere [27 (link), 34 (link)]. 25 μl reactions containing 1 μg of R-SSD3 peptide, in the presence and absence of 300 ng of either CK1 or CK2 or both containing 10 μCi 32P γ [ATP]. The reactions were performed in triplicate at 30°C for 1 hour, 4 hours, and 24 hours using one batch of radiolabeled ATP and enzymes from a single lot.
The reaction products were purified using SDS-PAGE, visualized by autoradiography, and band intensities were quantified using a Kodak 1D 3.6 imaging system. After autoradiography, the gel was overlaid on the autoradiogram, the individual protein bands were excised, and 32P incorporation was determined using a liquid scintillation counter (Beckman LS6500 system). To calculate the number of moles of phosphates transferred to 1 ug of peptide (0.466 pM) in the kinase reaction, the mean counts per minute obtained in the kinase reactions from triplicates (minus blank) were divided by the specific activity of the 32Pγ[ATP] in the kinase assay.
The reaction products were purified using SDS-PAGE, visualized by autoradiography, and band intensities were quantified using a Kodak 1D 3.6 imaging system. After autoradiography, the gel was overlaid on the autoradiogram, the individual protein bands were excised, and 32P incorporation was determined using a liquid scintillation counter (Beckman LS6500 system). To calculate the number of moles of phosphates transferred to 1 ug of peptide (0.466 pM) in the kinase reaction, the mean counts per minute obtained in the kinase reactions from triplicates (minus blank) were divided by the specific activity of the 32Pγ[ATP] in the kinase assay.