Figure 2.

Efficient encounter between LF and its target occurs from late endosomes. (A) CHO cells were incubated or not with 10 μM nocodazole for 2 h at 37°C (nocodazole was then present throughout the entire experiment), followed by 1 h at 4°C with 500 ng/ml PAn and 20 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of PNS was analyzed by Western blotting against PA, the NH₂ terminus of MEK1 (N) and LF. The amount of intact MEK1 (A) and MKK3 (B) was quantified by densitometry and normalized to the amount at time t = 0 (n = 4, errors bars represent SDs). (C) The kinetics of decrease in intact MEK1 were compared with that of decrease in MKK3. Western blots (inset) were quantified by densitometry as in A and B. (D) RAW 264 macrophages were incubated or not with 10 μM nocodazole (2 h at 37°C), followed by 1 h at 4°C with 500 ng/ml PAn and 100 ng/ml LF. 40 μg of PNS was analyzed by Western blotting against the NH₂ termini of MEK1 and MKK3. (E) HeLa cells were transfected or not with dominant-negative rab7N125I cDNA, incubated at 4°C for 1 h with 500 ng/ml PAn and 500 ng/ml LF and transferred to 37°C for different periods of time (in min) in a toxin-free medium. 40 μg of PNS was analyzed as in A (n = 4).