Figure 5. Flotillins are required for CT function in mammalian cells.

(A) Cos-1 cells were transfected with control or flotillin-1 (Flo-1) siRNA, and knockdown was gauged by immunoblot of crude cell lysates using antibodies against flotillin-1 and -2. (B) Cells treated as in A were incubated with buffer (no toxin) or 1 nM CT for the indicated times prior to analysis of intracellular cAMP by ELISA. Data (mean ± SEM; representative of 3 independent experiments) were normalized to forskolin response (~20% less forskolin-induced cAMP in flotillin-1 sRNA–treated cells as compared with controls; see text). (C) Cells transfected with control and flotillin-1 siRNA were incubated with 20 nM CT holotoxin for 90 minutes, and A1-chain retro-translocation was measured as in Figure 3E, except that a combination of CTA and CTB antibodies was used for immunoblot analysis (upper panel).