The RBM20 517–664 -V5 reporter was transfected into 293T cells with or without GFP-SRPK3. GFP-SRPK3/RBM20 517–664 -V5 co-expression resulted in RBM20 517–664 -V5 hyperphosphorylation (lanes 4 and 5), as indicated by a mobility shift that was abolished by incubation with lambda phosphatase (P, lane 6). U indicates untreated samples; N indicates control samples incubated without phosphatase. In the absence of the SRPK3 construct, a less pronounced but still noticeable mobility shift can be observed (lanes 1 and 2), consistent with RBM20 phosphorylation by endogenous kinases such as SRPK1, CLK1 or AKT2. Assay was performed in quadruplicate.
