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U.) per mg of proteins (mean values of n = 3 experiments �� SEM). B. HUVECs were either left untreated or intoxicated with LT (PA+LF, 3 + 1 ?g ml?1) for 24 h. Graph shows the distribution of annexin V-FITC-positive cells (x-axis) and propidium iodide positive cells (y-axis). http://www.selleckchem.com/screening/tyrosine-kinase-inhibitor-library.html Values correspond to the percentages of distribution of each cell population. C and D. Control immunoblots of LT activity on MEK2 cleavage. Western blot anti-actin shows equal protein loading. (C) HUVEC monolayers intoxication for 24 h with PA 3 ?g ml?1 alone or together with LF at various concentrations (?g ml?1). (D) HUVEC monolayers intoxication with LT (PA+LF, 3 + 1 ?g ml?1) or a catalytically inactive mutant LTm (PA+LFE687A 3 + 1 ?g ml?1). E. HUVEC monolayers were intoxicated 24 h with recombinant LT purified from E. coli (PA+LF, 3 + 1 ?g ml?1) or with B. anthracis purified native toxin (LT Ba: PA+LF, 3 + 1 ?g ml?1). F-actin was labelled with FITC-phalloidin and VE-cadherin with anti-cadherin5. Scale bar, 10 ?m. F. Caspase-3 activity in HUVEC monolayers intoxicated 24 h with recombinant LT purified from http://en.wikipedia.org/wiki/VAV2 E. coli (PA+LF, 3 + 1 ?g ml?1) or with B. anthracis purified native toxin (LT Ba: PA+LF, 3 + 1 ?g ml?1). zVAD 20 ?M was added to evaluate the specificity of caspase activation by LT. Activities are expressed as arbitrary units (A.U.) per mg of proteins (one representative experiment). G. Caspase-3 activity in HUVEC monolayers treated 24 h with a mix of MKK/MKI (10 ?M U0126, 20 ?M SB203580 and SP600125 10 ?M) or with LT (PA+LF, 3 + 1 ?g ml?1). Activities are expressed as arbitrary units (A.U.) per mg of proteins (mean values of n �� 2 experiments �� SEM). Immunoblots show the effect of LT and MKI on ERK phosphorylation (pERK) (inset). H. Gene transcript modulations assessed by qRT-PCR. HUVEC monolayers were intoxicated 24 h with B. anthracis purified native toxin (LT Ba: PA+LF, 3 + 1 ?g ml?1). Mean values of n �� 3 independent biological replicates per condition �� SEM (*P http://www.selleckchem.com/GSK-3.html at both 4 and 8 h. Genes found twice or more are indicated in bold. C and D. Immunoblots show the absence of impact of LT on the activation of Rac and Cdc42. HUVEC monolayers were intoxicated with LT (PA+LF, 3 + 1 ?g ml?1) for the indicated times. CNF1 toxin 10?9 M was used as positive control of Rho protein activation. Levels of active-Rac and active-Cdc42 were determined by GST-p21pak RBD pull down (RacGTP and Cdc42GTP).