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. 2012 Dec 31;288(6):3753–3767. doi: 10.1074/jbc.M112.415240

FIGURE 1.

FIGURE 1.

A, IP-MS identification of Trabid interactors and substrates. Immunoprecipitation (IP) of WT FLAG-Trabid and its C443S DUB-inactive version from transfected HEK293 cells. IPs, including an empty vector control, were resolved by SDS-PAGE and stained with Coomassie. Regions a–k of the gel were excised for MS and analyzed in the presence of isotopically labeled internal standards against various forms of polyubiquitin. The abundance of linkages measured (fmol) in select gel regions from WT and C443S (CS) IPs is displayed in the adjacent tables. The abundance reported for Lys-29 linkages represents the fraction of the signal detectable in the form of the K29-short (AK*IQDK) peptide and is an underestimate of the total Lys-29 signal. B, MS/MS analysis of the gel-resolved IPs identified peptide sequences matching several components of the STRIPAK complex, which co-eluted specifically with the Trabid IPs and not the vector control IP. The E3 ubiquitin ligase HectD1 was also identified as a specific Trabid-associated protein.