Figure 6

ARTD10 colocalizes with p62 bodies. A. GFP-ARTD10 and FLAG-Dcp1a, a P-body component, were co-expressed in COS7 cells. FLAG-Dcp1a was stained with a FLAG-specific antibody and co-localization of the two proteins was evaluated by confocal microscopy. B. GFP-ARTD10 and RFP-TIA1, a marker for stress granules, were co-expressed in COS7 cells and the localization evaluated. C. HA-ARTD10 and GFP-p62 were co-expressed in COS7 cells. HA-ARTD10 was stained with a HA-specific antibody and co-localization of the two proteins was evaluated. D. GFP-ARTD10 was expressed in COS7 cells. Endogenous p62 was stained with specific antibodies. Arrows indicate bodies that contain ARTD10 and are positive for p62. Double-arrows and arrowheads indicate p62 and ARTD10 only positive bodies, respectively. E. GFP-ARTD10 and mCherry-p62 were expressed in p62^−/− MEF and their subcellular localization evaluated. F. HA-ARTD10 and Myc-p62 were co-expressed in HEK293 cells. p62 and associated proteins were immunoprecipitated and analyzed by Western blotting. For control the expression of HA-ARTD10, Myc-p62, and actin was determined as indicated. G. COS7 cells expressing GFP-ARTD10 were stained for ubiquitin using mAb FK1, which recognizes ubiquitin polymers. H. As in panel F but with mAb FK2, which recognizes both ubiquitin monomers and polymers. I. GFP-ARTD10 and mCherry fusion protein with a p62 mutant unable to interact with ubiquitin (mCherry-p62ΔUBA) were expressed in p62^−/− MEFs. The localization of the proteins was determined. I. GFP-ARTD10 was expressed in p62^−/− MEFs and the cells were stained for ubiquitin using FK2.