GILZ is expressed upon stimulation with C3 exotoxin or toxin B.

<p>(A) HeLa cells were incubated with toxin B (50ng/ml) for indicated the time spans and immunoblots were performed from whole cell lysates using anti-Rac1 mAb102 recognizing only non-glucosylated Rac-1, and an anti-Rac1 mAb antibody recognizing total Rac1 as well as antibodies recognizing RhoB, actin or GILZ. (B) Immunoblot of GILZ and actin expression upon stimulation of HeLa cells with C2IN-C3lim (100 ng/mL) + C2IIa (200 ng/mL) for indicated time spans. (C) To explore the expression of additional GILZ isoforms, HeLa cells were transfected with 7.5 nM siRNA specific for GILZ or control siRNA for 48 h and subsequently either left untreated or stimulated with <i>C. difficile</i> toxin B (50 ng/ml) or 100 µM DEX for 4 h. Arrows mark three GILZ isoforms which were inhibited by the used GILZ siRNA. Note that only isoform 1 was induced by the used stimuli. (D) Cells were stimulated with toxin B for 2 or 4 h to determine <i>GILZ</i> mRNA expression by real-time RT-PCR. Mean + SD of 2 independent experiments normalized to untreated. (E) To assay transcriptional activity of the GILZ promoter cells were transfected with a luciferase reporter under control of a 2088 bp <i>GILZ</i> promoter and co-transfected with pCMV-ß-gal (for standardization) 24 h before infection with a <i>Y. enterocolitica</i> pYV⁺ and various mutant strains or treatment with DEX or toxin B. Means + SD of 4 independent experiments normalized to untreated. Significant differences compared to untreated are indicated by asterisks (p<0.05).