C3bot1E174Q-C2I is taken up into the cytosolic fraction of J774A.1 cells.

A. J774A.1 cells were incubated for 6 h at 37°C with C3bot1E174Q-C2I (4 µg/mL). For control, cells were incubated for the same time with either C2I (1 µg/mL)+C2IIa (2 µg/mL) or C3bot1E174Q-C2I (4 µg/mL)+C2IIa (8 µg/mL). For further control cells were left untreated or were incubated with C2I alone (4 µg/mL). Thereafter, cells were washed, and incubated for 5 min at 4°C with an antibody against C2I (1∶2,000) to remove non-internalized C2I or C3bot1E174Q-C2I. After an additional washing step, cells were incubated with digitonin (20 µg/mL in PBS) to extract the cytosolic proteins. The cytosolic fraction as well as the remaining cells were subjected to SDS-PAGE and characterized by Western blotting. Comparable amounts of total protein in the lanes were confirmed by Ponceau S staining. An antibody against the endosomal protein EEA1 was used to confirm that the cytosolic fraction did not contain early endosomal vesicles. B. C2I and C3bot1E174Q-C2I were detected with an antibody against C2I.