Protein Modification Analysis Workflow

After 2-DE, gels were either stained with colloidal Coomassie blue R solution (12% trichloroacetic acid, 5% ethanolic solution of 0.035% Serva blue R-250) or transferred onto PVDF membranes (Mini-PVDF transfer pack, Bio-Rad) in the Trans-Blot Turbo Transfer system (Bio-Rad) according to manufacturer’s instructions. For immunodetection of O-GlcNAc modified proteins, western-blot was performed with CTD110.6 antibody (1:5,000 dilution, Sigma-Aldrich) as already published^{28 (link)}. Phosphorylated proteins were detected using anti-phospho-Threonine antibodies (Q7 at 1:500, Qiagen) according to manufacturer’s protocol (Phosphoprotein Purification kit, Qiagen).