Fig. 8: SdeA induced serine ubiquitination inhibits VSVG trafficking through Golgi membranes.

A Confocal images showing the effect of SidE family effectors on VSVG trafficking during Legionella infection. A549 cells were transfected with VSV-G-GFP and cultured at 37 °C for 24 h to express the proteins before transferred to 40 °C. After 16 h incubation at 40 °C, cells were infected with Legionella for another 2 h then washed 3 times with PBS then moved to 32 °C for 0 min, 10 min, 20 min, 60 min, 90 min, 120 min to release VSVG from ER. A549 cells were fixed and VSVG trafficking was acquired with confocal microscopy after immunofluorescence staining. Scale bars, 5 μm. B Quantitative analysis of the effect of SidE family effectors on VSVG trafficking during Legionella infection. Colocalization between VSVG and GM130 was shown as Manders coefficient. Data represents 30 cells taken from 3 independent experiments. White boxes indicate insets which are split into red, green, blue channels and displayed on the right side of the image. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; data points are plotted as circles. C Western blotting analysis of the effect of SidE family effectors on VSVG trafficking during Legionella infection using EndoH. Upper bands indicate the EndoH resistant form (mature) and lower bands indicate the EndoH sensitive form of VSVG. D Quantification of (C) to indicate the effect of Legionella infection on the conversion of EndoH sensitive form to resistant form of VSVG upon 32 °C incubation. Data were analyzed with unpaired t test, ***P < 0.001, **P < 0.01, *P < 0.05.