Rngo binds to ubiquitin and to the proteasome and forms homodimers. (a) A schematic diagram of Rngo fusion proteins used in this study. Numbers corresponding to amino acid residues indicate domain boundaries and the extent of the introduced deletions. (b) The UBA domain of Rngo is required for binding to ubiquitin. Ubiquitin-conjugated agarose beads were used to pull down the MBP-Rngo fusion proteins indicated above each lane. MBP was used as a negative control. (c) Direct binding of Rngo to the proteasomal subunit Rpn10. FL MBP-Rpn10 was pulled down by GST–FL Rngo but not by GST alone. (d) Rngo binds to ubiquitin (Ub) and Rpn10 in vivo. Extracts of wild-type ovaries (left) and ovaries expressing Rngo-GFP (right) were used for co-IP experiments with the indicated antibodies. The anti-MBP antibody was used as a negative control. (e) Rngo forms homodimers via its RVP domain. S2 cells were cotransfected with FL Rngo-GFP and either FL or mutated versions of Rngo tagged with the HA epitope. Cell extracts were used for IP with an anti-GFP antibody. IP with an anti-GST antibody was used as a negative control. Untransfected cells and cells transfected only with the respective HA-tagged version of Rngo were used as additional negative controls. White lines indicate that intervening lanes have been spliced out.