Figure 2.

Suilysin-induced activation of GTPases in hBMECs was dependent on the concentration of cholesterol but not hemolytic activity. hBMEC cells were serum-starved and then treated with (A and B) S. suis supernatant or (C and D) suilysin protein at sublytic concentrations for indicated periods. (E) hBMEC cells pretreated with Y27632 or left untreated were then treated with suilysin that had been pretreated with cholesterol or left untreated. (F) hBMEC cells were treated with P353L mutant of suilysin at indicated concentrations. (A, C, E and F) RhoA and (B, D and F) Rac1 activation was detected using pull-down and Western blot assays with total RhoA and Rac1 amounts at each point in time as shown in the images of the endogenous control and representative samples. (C and D) Relative amounts of activated RhoA and Rac1 from three independent experiments with mean ± SEM (bottom panel)