FIG 1.

Reconstitution of mammalian DUBm. (A) Schematic of the mammalian SAGA DUBm. (B) Schematic representation of ATXN7 N-terminal fragments with 24 Q residues and 92 Q residues, where Q is glutamine. (C) Colloidal staining of DUBm subunits after elution with HA or Flag peptides from immunoaffinity agarose beads. The expression levels are similar for all the subunits analyzed, with the exception of the expression level for ATXN7-92Q NT. #, purified DUBm subunits in each lane. (D) Expression of exogenous Flag-ATXN7 NT in WCEs from Sf21 insect cells after cellular fractionation at 3 days postinfection in order to assess the solubility of ATXN7-24Q NT and ATXN7-92Q NT. Lanes S, supernatant (soluble fraction); lanes P, pellet (insoluble fraction). Numbers on the left are molecular masses (in kilodaltons). (E) Colloidal staining of the reconstituted DUBm with ATXN7-24Q NT or ATXN7-92Q NT after anti-HA (USP22) immunoprecipitation. (F) Immunoblot analysis of the reconstituted DUBm for which the results are presented in panel E. ATXN7-24Q NT or ATXN7-92Q NT, ATXN7L3, and ENY2 are all Flag tagged and are detected with anti-Flag antibody; HA-USP22 is detected with anti-HA antibody. *, modified forms of ATXN7-24Q NT or ATXN7-92Q NT due to ubiquitination (data not shown). Lanes M (C and E), molecular mass markers; IB, immunoblot analysis; IP, immunoprecipitation analysis.