Eea1 antibody

For colocalization studies of CedV F and mutants with early endosomes, antibody uptake assays were performed as described above with modifications. Briefly, at 24 h p.t., CedV F-expressing HeLa cells were incubated with the polyclonal rabbit anti-CedV F serum (1:200) for 1 h at 4 °C. After washing, cells were shifted to 37 °C for 5 or 30 min to allow endocytosis to occur. Then, surface-bound primary antibodies were blocked with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:50; LifeTechnologies, Darmstadt, Germany). After fixation with 2% PFA for 20 min and permeabilization with 0.2% Triton X-100 in PBS, a mouse anti-human early endosomal antigen 1 (EEA-1) antibody (1:50; BD Biosciences, Heidelberg, Germany) was added for 1 h at 4 °C for staining of early endosomes. Internalized primary rabbit antibodies were stained with AF 488-conjugated secondary antibodies (1:500; LifeTechnologies, Darmstadt, Germany). Primary mouse antibodies bound to EEA-1 were detected with AF 568-conjugated secondary antibodies (1:500; LifeTechnologies, Darmstadt, Germany). Cell nuclei were counterstained with 4′,6-Diamidin-2-phenylindol (DAPI). Representative images were recorded with a confocal laser scanning microscope (Leica SP5) and processed with the ImageJ software version 1.45 s [47 (link)].