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Activation of human or murine TLR4-MD2 by H. pylori LPS.

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posted on 22.12.2011, 00:10 by Thomas W. Cullen, David K. Giles, Lindsey N. Wolf, Chantal Ecobichon, Ivo G. Boneca, M. Stephen Trent

Activation of TLR4 was monitored using HEK-293 cells stably transfected with either human TLR4-MD2-CD14 or murine TLR4-MD2-CD14. TLR activation was monitored colorimetrically using a secreted alkaline phosphatase (SEAP) reporter gene placed under the control of an NF-κB inducible promoter. Highly purified LPS from the indicated strains of H. pylori was added to each well in triplicate at 0, 10, 100, 1000 and 10000 ng/ml. Values are the mean of results from triplicate wells ± standard deviation. When compared to wild type, LPS (10000 ng/ml) from the lpxE, lpxF, and lpxE/F mutants showed an ∼2, 6 and 10-fold significant increase in hTLR4-MD2 activation, respectively. hTLR4-MD2 acitvation by LPS prepared from the LpxR mutant and complemented strains were identical to that of wild type (p>0.05). Where appropriate, an asterisk was used to indicate significance (p ≤0.001) (A). For experiments with hTLR4-MD2, LPS from R. sphaeroides (1000 ng/ml) and E. coli (10 and 1000 ng/ml) was used as negative and positive controls for activation of hTLR4-MD2, respectively (B). When compared to wild type, LPS (10000 ng/ml) from the lpxE, lpxF, lpxR and lpxE/F mutants showed an ∼10, 8, 9 and 5 fold significant increase in mTLR4-MD2 activation, respectively. mTLR4-MD2 acitvation by LPS prepared from complemented strains were identical to that of wild type (p>0.05). Where appropriate, an asterisk was used to indicate significance (P ≤0.01) (C). For experiments with mTLR4-MD2, endotoxin free water and LPS purified from E. coli (10 and 1000 ng/ml) was used as negative and positive controls for activation of mTLR4-MD2, respectively (D). LPS from R. sphaeroides is a known mTLR4-MD2 agonist and was not used as a negative control. All results agreed between experimental and biological replicates. Cells were stimulated for 24 hours with the indicated ligands.

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