Fig. 5: Identification of GRASP55 ubiquitination with mass spectrometry.
From: Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection
A In vitro reaction of GRASP55 ubiquitination by SdeA for mass spectrometry analyses. In total, 20 μg purified GRASP55 was incubated with SdeA and ubiquitin in the present of NAD. 10% reaction products were separated with SDS-PAGE and then stained with Coomassie blue or blotted with with antibodies against ubiquitin, GRASP55 to check the ubiquitination, the rest samples were subjected to mass spectrometry analyses. B Spectrum of GRASP S408-ubiquitin cross-linked peptide. C Validation of of ubiquitination sites in GRASP55. C-terminally HA-tagged wild-type and GRASP55 mutant were co-expressed with SdeA in HEK293T cells. After 24 h the cells were lysed for HA immunoprecipitation. Purified GRASP55-HA proteins were separated with SDS-PAGE followed by blotting using anti-HA and anti-ubiquitin antibodies. D Ubiquitination assay of wild type GRASP55 and mutant in cells infected with Legionella.
