Figure 6. EMC6 is associated with the RAB5A-BECN1 complex. (A, B, D, E and H) Direct or indirect immunofluorescence analysis by confocal microscopy was performed on cells, and nuclei were stained with H33342 as indicated. (A) U2OS cells were cotransfected with EMC6- and GFP-ZFYVE1-expressing plasmids for 24 h and then stained with an anti-EMC6 antibody. (B) U2OS cells were cotransfected with plasmids expressing FLAG-EMC6 and GFP-BECN1 for 24 h and stained with an anti-FLAG antibody. (C) GST-EMC6 fusion protein and the GST protein immobilized on Glutathione-Sepharose beads were incubated with GFP-BECN1-transfected HCT116 cell lysates at 4°C for 4 h. GFP and GST were detected in the washed beads by western blot. (D) U2OS cells were cotransfected with plasmids expressing FLAG-EMC6 and GFP-RAB5A for 24 h and then stained with an anti-FLAG antibody. (E) U2OS cells were cotransfected with plasmids expressing EMC6-GFP and DsRed-RAB5A for 24 h. (F) HCT116 cells were transfected with a plasmid expressing FLAG-EMC6 for 24 h. Total cell extracts were subjected to IP using either an anti-FLAG or a nonspecific control mIgG, as indicated. RAB5A was detected in the IP proteins by western blot. (G) GST-EMC6 fusion protein and the GST protein immobilized on Glutathione-Sepharose beads were incubated with GFP-RAB5A-transfected HCT116 cell lysates at 4°C for 4 h. GFP and GST were detected in the washed beads by western blot. (H) The GST-EMC6 fusion protein or the GST protein immobilized on Glutathione-Sepharose beads were incubated with His-RAB5A or His-RAB5A loaded with GDP or GTPγS. His-RAB5A and GST were detected in the washed beads by western blot. (I) U2OS cells were transfected with FLAG-EMC6 for 24 h and then stained with anti-EMC6 or anti-EEA1 antibody.