WASp associates with SUMO-pathway proteins in vivo. (A) LC-MS/MS. Number of unique polypeptides of WASp-associated, SUMO-pathway proteins isolated from nuclear and cytosolic fractions of TH1-skewed, primary TH cells (reports on endogenous WASp-proteome) or Jurkat TH cells (reports on Flag/Myc-tagged transfected WASp-proteome) by immunoprecipitation (IP) with anti-WASp antibody (Ab) or anti-FLAG/Myc antibodies (2-step immunopurification, 1stIP: Flag Ab, 2ndIP: Myc Ab). MultiConsensus reports of peptides/proteins were generated from 3 to 4 biological replicates after applying the filtering criteria previously described15 (supplemental Figure 1). (B) Validation of MS-generated WASp-SUMO associations by coIP. Protein complexes isolated by reciprocal and sequential coIP (eg, WASp>RanBP2 sequence denotes 1stIP: WASp, 2ndIP: RanBP2) from whole cell extracts of TH1-skewed, human primary TH cells resolved by western blotting the same gel with indicated antibodies. NEM−, nontreated with NEM.
