Ubiquitination of Bacterial Effector SidH

2 µM SidH WT or SidH HM, 1 µM UBE2D2, 1 µM GST-Lubx, 0.3 µM E1, 12 µM ubiquitin and 2.5 mM ATP were incubated for 1 h at 30 °C. Samples were loaded onto 4–20% Tris glycine gradient gels (Biorad) and analysed by Coomassie staining or western blot using either Ubiquitin P4D1 (sc-8017, SantaCruz) or GST (sc-138, SantaCruz) antibody in a solution containing 5% BSA, 0.2% Tween20 and PBS. Following numerous washing steps with 0.2% Tween20 in PBS, blots were incubated with IRDye® 800CW Donkey anti-Mouse IgG Secondary Antibody (Li-Cor) for 1 h at room temperature and visualized by fluorescence. Following SDS-P AGE and coomassie staining, the gel streak corresponding to ubiquitinated SidH were excised and subjected to in-gel digestion with trypsin to generate peptides containing the Lys-ε-Gly-Gly (diGLY) remnant. The peptides were introduced into the Orbitrap Fusion Lumos (Thermo Scientific, SanJose) mass spectrometer. Data analysis was done with MSFragger v3.7^{41 (link)}. Carbamidomethyl (C) was set as fixed modification, Acetyl (Protein N-term), Oxidation (M) and GlyGly (K) as variable modifications.