Dyrk1a 7d10

Cells were lysed in TENT buffer (50 mM Tris, pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100) supplemented with 2 mM NaF, 2 mM NaVO₃, 2 mM PMSF, and 1× cOmplete protease inhibitor cocktail (Roche) for 30 min on ice. Insoluble debris was pelleted by centrifugation at 21,000 g for 10 min at 4°C. Lysates were denatured in LDS sample loading buffer (Life Technologies) at 100°C for 5 min, and electrophoresed on 4–12% Bis-Tris gradient gels (Life Technologies). Proteins were transferred to PVDF membranes and probed with primary antibodies for: DYRK1A (7D10; Abnova), Cyclin D3 (C-16; Santa Cruz Biotechnology, Inc.), Cyclin D2 (M-20; Santa Cruz Biotechnology, Inc.), phospho-RB S807/811 (D20B12; Cell Signaling Technology), phospho-Src family Y461 (D49G4; Cell Signaling Technology), phospho-PDK1 S241 (C49H2; Cell Signaling Technology), Lyn (5G2, Cell Signaling Technology), phospho-NFATc2 S326 (sc-32994; Santa Cruz Biotechnology, Inc.), and FLAG (M2; Sigma-Aldrich) and detected with HRP-conjugated secondary antibodies and ECL substrate (GE Healthcare). β-Actin was detected using an HRP-conjugated primary antibody (C4; Santa Cruz Biotechnology, Inc.). Band densitometry values were calculated using ImageJ software.