Secretional Blockade by VSL#3 Figure 3. L. casei-induced inhibition of IP-10 secretion is mediated by a post-transcriptional mechanism (A) Mode-K cells were stimulated with TNF (10 ng/ml) and L. casei (moi 20) for 15 and 30 min and TNF-induced proteasomal degradation of IkB and (B) TNF-induced activation of RelA was analyzed by Western Blot in two independent experiments. (C) TNF-induced recruitment of NFkB RelA to the IP-10 promoter was analyzed in a ChIP experiment using an anti-RelA antibody after pre-incubation of Mode-K cells with L. casei (moi 20) (1h) and subsequent stimulation with TNF (10 ng/ml) for 2 h. (D) Control (ctr-vector)- and IP-10 reporter (p-IP-10)-plasmid transfected Mode-K cells were stimulated with TNF (10 ng/ml) with or without L. casei (moi 20) and intracellular luciferase activity was measured by a luminescence assay after 24 h. (E) Mode-K cells were stimulated with TNF (10 ng/ml) and/or L. casei (Moi 20) for 6 h followed by mRNA isolation, RT-PCR and qPCR. (F) Mode-K cells were stimulated with TNF (10 ng/ml) alone and together with L. casei (moi 20) for 24 h. Intracellular accumulation of IP-10 was analyzed by Western blot and the shown figure is representative for more than three independent experiments. doi:10.1371/journal.pone.0004365.g003 PLoS ONE | www.plosone.org 6 February 2009 | Volume 4 | Issue 2 | e4365