FIGURE 5.

RAP80 is an in vivo target of BRCC36 Lys⁶³-specific DUB activity. A, FLAG-IP of ectopic BRCC36 complexes was performed and probed with anti-BRCC36 and anti-RAP80 antibodies as indicated. The anti-BRCC36 antibody (J86) recognizes the C-terminal 20 amino acids of BRCC36. C-terminal epitope tags strongly reduce J86 recognition on Western blot, accounting for the weak signal of BRCC36 species with C-terminal FLAG-HA tags (C-B36 and C-QSQ) compared with N-terminal FLAG-HA-tagged BRCC36 (N-B36 and N-QSQ). B, RAP80 is ubiquitinated by both WT and K48R mutated ubiquitin. 293T cells expressing FLAG-HA-RAP80 (RAP80) were mock-transfected or transfected with either WT ubiquitin or ubiquitin K48R mutant containing an N-terminal 6-histidine sequence. Ubiquitinated proteins were purified over a Ni²⁺-agarose column and eluted with imidazole-containing buffer. IB was performed with an anti-HA antibody. C, Ubc13 knockdown strongly decreases the polyubiquitinated form of RAP80 associated with BRCC36 QSQ. FLAG-IP of ectopic BRCC36 QSQ complex was performed following control (Ct) or Ubc13 siRNA-mediated knockdown. IB was performed on FLAG-purified BRCC36 complexes as indicated. D, FLAG and HA tandem immunoaffinity purification was performed for ectopic BRCC36 QSQ from HeLa S3 nuclear extracts. The RAP80-Ub2 Coomassie-stained band was excised and trypsin-digested prior to mass spectrometry analysis. M, molecular weight marker; Mock, mock-transfected cell line. E, peptide sequences obtained by mass spectrometry of tryptic digests from the RAP80-Ub2 band. K#, RAP80 lysine residues that are conjugated to ubiquitin. A peptide sequence indicating the presence of Lys⁶³-linked polyubiquitin was also detected by mass spectrometry from tryptic digests of the RAP80-Ub2 band. F, IB of ectopically expressed FLAG-HA BRCC36 or FLAG-HA BRCC36-QSQ after FLAG-IP. Increased RAP80 polyubiquination, specifically Lys⁶³-linked ubiquitin, is observed in association with BRCC36-QSQ compared with BRCC36.