Fig. 6: Serine ubiquitination impairs GRASP55 function.
From: Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection
A Assay of the effect of PR-ubiquitination on GRASP55 dimerization in vitro. GRASP55-GFP purified from HEK293T cells were modified in vitro using SdeA and ubiquitin in the presence of NAD+, ubiquitinated GRASP55-GFP was then incubated with purified His-tagged GRASP55. Interaction between differently tagged GRASP55 proteins were analyzed with co-immunoprecipitation followed with western blotting. B Assay of the effect of PR-ubiquitination on GRASP55 dimerization in vivo. HA-tagged GRASP55 and GFP-tagged GRASP55 serine mutant were co-expressed with SdeA in HEK293T cells. Protein interaction between differently tagged GRASP55 were analyzed with co-IP and western blotting. C Confocal images showing that GRASP55 mutant is resistant to Golgi fragmentation caused by SdeA expression. Golgi areas of more than 60 cells from 3 replicates of each condition were measured with ImageJ software. Scale bars, 10 μm. D Data are shown as means ± SEM of more than 70 cells taken from three independent experiments. Data were analyzed with unpaired t test, ***P < 0.001.
