Interactions of SRPK1 and SRPK2 with Cp.

(A) In vitro GST pull-down assay was performed using recombinant GST-tagged CpY132A with His-tagged SRPK1WT or SRPK2WT. The result was analyzed by SDS-PAGE. Both SRPK1 and SRPK2 bind Cp directly. (B) Microscale thermophoresis (MST) analysis to measure the binding affinity between GST-CpY132A and SRPK1WT or SRPK2WT. Kd of 83.6 ± 19.8 nM and 101.1 ± 15.9 nM were determined for the interactions of GST-CpY132A with SRPK1WT (left panel) and SRPK2 WT (right panel), respectively. Values are mean ± S.D. for three independent experiments. (C) In vitro radioactive phosphorylation assays of purified CpY132A. Recombinant GST-CpY132A was phosphorylated by SRPK1 or SRPK2 using [32P]ATP. Reactions were quenched by SDS loading buffer at the indicated time points and the reactions were resolved by SDS-PAGE followed by autoradiography. SRPK2 phosphorylated Cp more effectively than SRPK1 under the same condition. Experiments have been repeated three times and the representative data are shown.