Molecular Mimicry by an F-Box Effector of Legionella pneumophila Hijacks a Conserved Polyubiquitination Machinery within Macrophages and Protozoa (original) (raw)
Figure 6
The F-box AnkB protein mimics host F-box proteins by interacting with host SKP1 in vivo and SKP1 is essential for intracellular bacterial proliferation.
A) In vivo co-immunoprecipitation of AnkB and SKP1 in co-transfected cells. HEK-293 cells were co-transfected with plasmids encoding 3X-Flag AnkB, AnkB9L10P/AA, BAP or SKP2 and SKP1-GST. Total protein input in total cell lysate was equivalent in all the lanes (not shown). Cell lysates were purified using anti-flag resin and the resulting supernatants were analyzed by immunoblotting with antibodies against Flag and GST. B) Representative confocal images of HEK-293 cells that were untreated or transfected with RNAi followed by infection and labeling of the bacteria at 16 h to score replicative vacuoles. C) Quantitative analyses of the formation of replicative vacuoles were based on examination of 100 infected cells. The different color bars represent the number of bacteria/vacuole. The data represent analyses of the mean and standard deviations of 100 infected cells, and are representative of two independent experiments. D) Knockdown expression of SKP1 protein by siRNA silencing in HEK-293 cells was confirmed by Western blot of HEK-293 cell lysates probed with an anti-SKP1 antibody. The blots were re-probed with anti-actin antibodies, which showed equivalent quantities of proteins were loaded onto the gel. The results are representative of three independent experiments.