FIG. 6.
CNF entry is dependent on endosomal acidification. (A) HeLa cells were preincubated with bafilomycin A1 (BafA1) (100 nM) for 1 h at 37°C. Afterwards, cells were intoxicated with GST-CNF1 (1 nM) and GST-CNFY (1 nM) overnight at 37°C in the presence of bafilomycin A1. A Rho shift assay was performed after lysis of the treated cells. (B and C) To investigate CNFY translocation through the plasma membrane by an acidic pulse, HeLa cells were preincubated with bafilomycin A1 (100 nM) at 37°C for 45 min. Cells were then washed with HBSS (pH 7.5), and 1 μg/ml GST-CNF1 or GST-CNFY was bound to the cells for 2 h at 4°C. Thereafter, cells were washed with HBSS (pH 7.5), HBSS (pH 5.2), or HBSS (pH 4.8) and incubated at 37°C for the times indicated using these different pH values. Afterwards, cells were further incubated overnight at 37°C in DMEM in the presence of bafilomycin A1 (100 nM). Translocation of CNF molecules through the plasma membrane into the cytosol was monitored by morphological changes of the cells (B) or by the Rho shift assay (C).