C. jejuni -triggered Cdc42 activation is time-dependent and requires intact lipid rafts. (A) Quantification of Cdc42 activity during the course of infection. INT-407 cells were infected with wt C. jejuni 81-176 for indicated periods of time. The presence of active Cdc42-GTP was quantified by G-Lisa and GST-CRIB pulldown. One hundred % of GTPase activity corresponds to the highest amount of detected Cdc42-GTP level (right lane). Similar quantities of total Cdc42 and GAPDH were confirmed by Western blotting. (B) Effect of Cdc42 expression knockdown on C. jejuni invasion. INT-407 cells were transfected with Cdc42-siRNA as well as a scrambled siRNA as control. After 48 hours, cells were infected with C. jejuni for 6 hours. Intracellular bacteria were quantified by gentamicin protection assays. Immunoblotting with α-Cdc42 antibody confirmed down-regulation of the protein. GAPDH expression levels were determined as control. (C) Effects of MβCD targeting lipid rafts on host cell internalization of C. jejuni. INT-407 monolayers were pre-incubated with the indicated concentrations of MβCD for 30 min, followed by 6 hours infection with wt C. jejuni 84-25. Intracellular C. jejuni were quantified by gentamicin protection assays. The presence of active Cdc42-GTP was analyzed by CRIB-GST pulldown and quantified. One hundred % of activity corresponds to the highest amount of detected Cdc42-GTP level (lane 2). Similar quantities of total Cdc42 and GAPDH were confirmed by Western blotting. (*) P ≤ 0.05 and (**) P ≤ 0.005 were considered as statistically significant as compared to the control.