FIGURE 4.

Protein-protein interactions with MPN⁻ domain proteins are a common mechanism of regulating JAMM/JAB DUB activity. A, IB of ectopic Poh1 complexes after FLAG-IP from 293T cells. FLAG-HA-tagged plasmids encoding Poh1 were co-transfected with control (Ct) or Rpn8 siRNA, and IP was performed 48 h later (top). Depletion of Rpn8 from Poh1 complexes abrogates Poh1 DUB activity (bottom). Equal amounts of FLAG peptide-eluted Poh1 complexes were incubated with hexa-K63-Ub for 1 h, and the products were detected by IB with the P4D1 antibody to ubiquitin. B, IB of ectopic BRCC36 complexes after FLAG-IP from HeLa S3 cell nuclear extracts. Cells were treated with control or Abraxas siRNA, and IP was performed on nuclear extracts 48 h later (top). Abraxas deficiency abrogates BRCC36 DUB activity from nuclear extracts (bottom). Equal amounts FLAG peptide-eluted BRCC36 protein were incubated with hexa-K63-Ub for 1 h, and the products were detected by IB with the P4D1 antibody. C, IB of ectopic BRCC36 complexes after FLAG-IP from HeLa S3 cell nuclear extracts. Cells were treated with control or BRCC45 siRNA, and IP was performed on nuclear extracts 48 h later (top). BRCC45 deficiency abrogates BRCC36 DUB activity from nuclear extracts (bottom). Equal amounts of FLAG peptide-eluted BRCC36 protein were incubated with hexa-K63-Ub for 1 h, and the products were detected by IB with the P4D1 antibody.