2.4. In Vitro Kinase Assays

Kinase assays were carried out at 30 °C in a total volume of 25 μL containing 12 mM Hepes pH 7.5, 10 mM MgCl₂, 25 μM ATP, 1–3 μg of the appropriate substrate (GST-LBRNt(62–92) or GST-TAF15 or His-TAF15), and 1–2 μg GST-SRPK1. For the inhibition assays, GST-SRPK1 was incubated with GST-LBRNt(62–92) under the assay conditions in the presence of increasing concentrations of GST-TAF15, GST-TAF15-RGG, and GST-TAF15(1–320) or Nucl, SAFA, and A2B1 peptides, as indicated. Nucl (RGGGRGGFGGRGGGRGGRGGFGGRGRGGFGGRGGFRGGRGG), SAFA (RGGGHRGRGGFNMRGGNFRGGAPGNRGGY) and A2B1 (RGGNFGFGDSRGGGGNFGPGPGSNFRGG) peptides, comprising the RGG repeats of nucleolin, HNRPU (SAFA), and HNRNPA2B1, respectively, were provided by GeneCust, Boynes, France. The samples were incubated for 30 min, and the reaction was stopped by adding 6 μL of 5 × SDS sample buffer and heating at 95 °C for 3 min. Phosphoproteins were detected via autoradiography using Super RX (a Fuji medical X-ray film), and signals were quantified by excising the radioactive bands from the gel and scintillation counting. p-values were determined using a two-tailed, unpaired student’s t-test.