Molecular Mimicry by an F-Box Effector of Legionella pneumophila Hijacks a Conserved Polyubiquitination Machinery within Macrophages and Protozoa (original) (raw)
Figure 5
Ectopically expressed 3X-Flag AnkB is distributed at the periphery of HEK-293 cells, co-localizes with the three SCF components and poly-ubiquitinated proteins, and restores intracellular growth to the ankB mutant.
A) Representative confocal images of HEK-293 cells transfected with plasmids encoding 3X-Flag BAP or 3X-flag AnkB. At 24 h following transfection, cells were labeled using a mouse anti-Flag (green) and WGA conjugated to Alexa Fluor-647 (red). Nuclei were stained with DAPI (purple). The white arrow indicates peripheral localization of 3X-Flag AnkB. B) Representative confocal images of HEK-293 cells that were co-transfected with plasmids encoding 3X-flag AnkB or BAP and SKP1-GST. Cells were stained with anti-Flag (green) and anti-GST (red) and nuclei were visualized with DAPI (blue). Arrows indicate peripheral co-localization of AnkB and SKP1. C) 3X-Flag AnkB co-localizes with polyubiquitinated proteins at the cell periphery. At 24 h following transfection cells were labeled using a rabbit anti-flag antibody (green) and a mouse anti-polyubiquitin antibody (red). Nuclei were labeled with DAPI (blue). The white arrow indicates heavy co-localization of 3X-Flag AnkB with polyubiquitin. D) Representative confocal images at 12 h post-infection of HEK-293 cells transfected for 24 h with a plasmid encoding 3X-Flag AnkB. The cells were labeled with an anti-Lpn antibody (green) and an anti-Flag antibody (red). The data are representative of three independent experiments.