Aida image analyzer 5.0 program

The PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, Beverley, MA, USA), which is a reconstituted E. coli-based in vitro transcription-translation system, was used to express a DYRK1A construct comprising the catalytic domain of DYRK1A fused to an N-terminal Strep-tag II sequence. Reactions were run in a total volume of 10 μL with 10 ng/μL pET-ST2-DYRK1A [9 (link)] and 80 ng/μL SF3B1-NT-His6 as a substrate [51 (link)] at 37°C for 1 h. AnnH75 or solvent control (3% DMSO) was added as indicated. Reactions were stopped by adding 2x Laemmli sample buffer and 5 mM EDTA. Tyrosine autophosphorylation of DYRK1A and phosphorylation of SF3B1 on Thr434 was analysed by western blotting. Band intensities were quantified using the AIDA Image Analyzer 5.0 program (Raytest, Straubenhardt, Germany).

The inhibitors were added to the cultured cells from stock solutions in DMSO to the desired final concentration. For cytotoxicity assays, HeLa cells were grown in 96-well plates (20,000–30,000 cells per well) and incubated with the test compounds for 3 days before cell viability was evaluated with the help of a tetrazolium dye assay (XTT assay, AppliChem GmbH, Darmstadt, Germany).
Inhibitor assays of endogenous DYRK1A activity were performed with overexpressed GFP-SF3B1-NT as described previously [21 (link)]. Briefly, HeLa cells were grown in 6-well plates and treated with test compounds for 18 h before cells were lysed with 100 μL SDS lysis buffer (20 mM Tris HCl pH 7.4, 1% SDS) at 96 °C and sonicated. Total cellular lysates were analysed by immunoblotting with a custom-made rabbit antibody for detecting phosphorylated T434 [43 (link)] and a goat antibody for GFP (no. 600-101-215, Rockland Immunochemicals, Gilbertsville, PA, USA). pT434 signals were quantified using the AIDA Image Analyzer 5.0 program (Raytest, Straubenhardt, Germany). Relative phosphorylation of SF3B1 was calculated by normalisation to total protein levels as determined from GFP immunoreactivity. IC₅₀ values were determined by non-linear curve fitting using the GraphPad Prism 5.0 program (GraphPad Software, La Jolla, CA, USA).