FIGURE 2.

Proteomic profile of the WTAP complexes. A, the domains and unique amino acid repeats in the Hakai protein. Various Hakai deletion mutants are shown. The interaction of these mutants with WTAP was assessed by means of co-immunoprecipitation (IP) experiments. B, the cellular localization of Hakai-V5 and Hakai-delRING-V5. The nucleus was stained with TO-PRO-3 reagent. Bar, 10 μm. C, immunoprecipitation of Hakai complexes from the tetracycline-inducible HEK293 stable cell lines expressing Hakai-V5 or Hakai-delRING-V5. Immunopurified Hakai and its interacting proteins were resolved by SDS-PAGE and stained with SYPRO Ruby solution. The efficiency of immunoprecipitation was determined by Western blot. D, the proteomic profile of the WTAP complexes. This list shows the proteins isolated using the H1122, H1137, or Y6828 antibodies with a unique peptide number of ≥3 from HUVEC extracts or from PFA(+) HeLa cell extracts but not with the negative control antibody K7124 from HUVEC extracts or the anti-V5 antibody from the Hakai-delRING-V5 sample. The complete list of the identified proteins is presented in supplemental Table S1. The values represent the unweighted spectrum count (SPC) level divided by the molecular weight (MW) in kDa to determine the relative quantity of the immunopurified proteins. Hierarchical clustering was performed using JMP 7 software (SAS Institute, Cary, NC). E, immunopurification of the proteins cross-linked to WTAP. HeLa cells were cross-linked with paraformaldehyde, and the proteins cross-linked to WTAP were immunopurified with the H1122 antibody. Interacting proteins were resolved by SDS-PAGE and stained with SYPRO Ruby solution.