Bacterial Effector Binding to Ribosomal Protein S3 Subverts NF-κB Function
Figure 5
NleH1 reduces the nuclear abundance of RPS3.
A. Immunoblot analysis of cytoplasmic and nuclear fractions of 293T cells transfected with NleH1, NleH2, or an HA-epitope control, in the presence or absence of TNF-α (100 ng/ml) stimulation for 1 h. Blots were probed with α-RPS3, α-p65, α-HA, α-tubulin, and α-PARP monoclonal antibodies. B. Quantification (n≥4) of the fold-increase in nuclear RPS3 as assessed by densitometry analysis of immunoblots in the absence (open bars) or presence (black bars) of TNF-α stimulation. RPS3 signal intensity was normalized to tubulin (cytoplasmic) and PARP (nuclear). Asterisks indicate significantly different compared with HA transfection (p<0.05, ANOVA). C. Quantification of the fold-increase in nuclear RPS3 following a 3 h infection of HeLa cells with E. coli O157:H7 EDL933 strains possessing or lacking nleH1 and/or nleH2, as well as with strains complemented with the indicated NleH plasmids (n = 3). Asterisks indicate significantly different compared with wild-type infection (p<0.05, ANOVA). D. Immunoblot analysis of IκBα phosphorylation induced by TNF-α (left) or PMA (right), and total IκBα, in the presence or absence of NleH. E. Immunoblot analysis of the impact of C. rodentium NleH and EHEC NleH1 truncations and site-directed mutants on RPS3 nuclear abundance. Blots were probed with α-HA (top), α-RPS (middle), and α-PARP (bottom) monoclonal antibodies. The HA-input panel depicts the expression levels of the indicated constructs. The α-RPS panel depicts the nuclear abundance of RPS3 after stimulation with TNF-α in 293T cells transfected with the indicated constructs. The α-PARP signal was used for normalization of immunoblot signal intensities. F. Quantification (n = 3) of the fold-increase in nuclear RPS3 as assessed by immunoblotting in the presence of the indicated NleH expression plasmids. Asterisks indicate significantly different compared with NleH1 transfection (p<0.05, ANOVA).