GEF-H1 mediates RhoA activation during <i>S. flexneri</i> cell invasion.

<p>(A) Western blot analysis of subcellular distribution of GEF-H1 and cingulin upon <i>S. flexneri</i> invasion of MDCK cell monolayer. Densitometric analysis of GEF-H1 and cingulin in soluble and insoluble protein fractions during <i>Shigella</i> infection. Bars represent mean±SD <i>(* p<0.01 compared to the rest of time points)</i>. (B) Real-time PCR assessment of GEF-H1 expression in response to <i>S. flexneri</i> invasion of MDCK cells. Bars represent mean±SD <i>(* p<0.01 compared to non-infected cells)</i>. (C) GST pull down assays to assess activation of small GTPases in response to GEF-H1 expression. HEK293 cells were transfected with GEF-H1, RhoA, Rac1 and Cdc42 expression vectors, GEF-H1 (Y395A) mutant and constitutive active mutants for Rac1 (G12V) and Cdc42 (G12V). (D) GST pull down assays to assess activation of RhoA to <i>S. flexneri</i> infection. HEK293 cells were transfected with GEF-H1 mutant (Y395A) defective in nucleotide exchange or the empty plasmid as a control and incubated with <i>S. flexneri</i> for 10 minutes. Expression of GEF-H1 (Y395A) decreased baseline activation and prevented the rapid activation of RhoA during <i>Shigella</i> invasion of HEK293 cells. (E) NF-κB activation in response to <i>S. flexneri</i> invasion of HEK293 cells in the absence or presence of dominant negative GEF-H1 (Y395A) mutant. Bars represent mean±SD <i>(* p<0.05 compared to control)</i>. (F) Gentamicin protection assays of <i>S. flexneri</i> infection of HEK293 cells transfected with GEF-H1 (Y395A) or dominant negative RhoA (T19N) expression vectors. Bars represent mean±SD <i>(* p<0.05 compared to control plasmid)</i>. (G) Gentamicin protection assays of <i>S. flexneri</i> infection of HEK293 cells in the presence of two different GEF-H1 siRNAs. Bars represent mean±SD <i>(* p<0.05 compared to control siRNA)</i>.