Fig. 11. Exotoxin C3 blocks HIF-1 mRNA and protein overexpression and VEGF mRNA stimulation. Cells were pre-treated with 50 µg/ml of C3 toxin for 24 hours in normoxia. After this, cells were placed in hypoxia (H) or kept in normoxia (N) for 4 hours. Cells were fractionated into soluble and membrane fractions and proteins were separated by SDS-PAGE. Efficiency of ADP-ribosylation by C3 toxin was verified by immunodetection of RhoA, as the ADP-ribosylation reduces protein mobility (A). Alternatively, following incubation with the toxin, cells were exposed to hypoxia or in normoxia for 4 hours and total RNA isolated. The effects of the toxin were evaluated on mRNA levels (B) of HIF-1, VEGF and -tubulin as the negative control, and on protein expression (C) of HIF-1 and -tubulin by RT-PCR or western blot analysis. These experiments were carried out at least twice. Significant differences (P<0.05) from normoxia are indicated by asterisks (*).