Sg 77

Protein samples were denatured with NuPAGE LDS sample buffer containing NuPAGE sample reducing agent and boiled at 70 °C for 10 min prior to use. Proteins were separated by gel electrophoresis using NuPAGE Novex 4–12% Bis-Tris precast gels and transferred onto PVDF membranes using the iBlot gel transfer device (Thermo Fisher Scientific). For dot-blot assays, protein samples were spotted on nitrocellulose membranes (88018, Thermo Fisher Scientific) and dried at room temperature. Membranes were blocked with 5% non-fat milk in 0.2% Tween 20 phosphate buffered saline (PBS) for 30 min at room temperature and probed overnight at 4 °C with the following primary antibodies: mouse monoclonal anti-phosphoepitope SR proteins (1:500, MABE50, Milipore), rabbit anti-HA Tag (1:250, SG77, Invitrogen), mouse monoclonal anti-DYRK1A (1:200, sc-100376, Santa Cruz). Next, membranes were washed and incubated with StarBright Blue 700 goat anti-mouse IgG secondary antibody (1:2500, 12004159, Bio-Rad), StarBright Blue 520 goat anti-rabbit IgG secondary antibody (1:2500, 12005869, Bio-Rad) and hFAB rhodamine anti-tubulin antibody (1:2500, 12004165, Bio-Rad) for 1 h at room temperature. Immunoreactive bands were visualized in a ChemiDoc MP gel imaging system (Bio-Rad). Densitometric analysis was performed using Fiji (ImageJ) software [22 (link)].