Figure 3.

Alteration of the dynamics of late endosomal intraluminal vesicles leads to a delay in MEK1 cleavage by LF. (A) CHO cells were incubated or not for 18 h with the anti-LBPA antibody 6c4 (50 μg/ml), washed, further incubated at 4°C for 1 h with 500 ng/ml PAn and 250 ng/ml LF, and transferred to 37°C for different periods of time (in min) in a toxin-free medium. 20 μg of PNS was analyzed by Western blotting to detect PA^heptamer, the NH₂ terminus of MEK1 and LF. The amount of intact MEK1 was quantified as in Fig. 2 A (n = 4). (B) HeLa cells were transfected with siRNA against ALIX, the efficiency of which was examined 78 h later by Western blotting using antibodies against ALIX. Cells were then incubated at 4°C for 1 h with 500 ng/ml PAn and 100 ng/ml LF, transferred to 37°C for different periods of time (in min) in a toxin-free medium, homogenized, and 20 μg of PNS was analyzed to detect the presence of PA^heptamer and the NH₂ terminus of MEK1. Quantifications were performed as in A. (C) HeLa cells were treated as in B. 80 μg of PNS were centrifuged at 100,000 g for 1 h and the pellet analyzed by Western blotting for LF. (D) CHO cells were treated as in A and incubated with 500 ng/ml PAn and 500 ng/ml FP59. 20 μg of cell lysates were loaded on native or SDS-PAGE before Western blotting against EF-2. (E) ALIX was knocked down in HeLa cells as in B, incubated with 500 ng/ml PAn with 500 ng/ml FP59, and the modification of EF2 analyzed as in D.