Figure EV4. The ^{mC enp‐ F}R2687E mutant interacts, likely in a non‐specific manner, with full‐length Nup133 but not with its C‐terminal domain and localizes in GLFG bodies.

• A, B
  Y2H interactions between LexA alone (−) or fused to mNup133 full‐length (FL, [aa 1–1,155]) or C‐terminal domain (CTD, [aa 501–1,155]) and GAL4‐AD either alone (−) or fused to mCenp‐F‐SID (WT or bearing the indicated mutations) or Nup107 [aa 784–925] were assayed based on growth on ‐LWH medium supplemented, when indicated, by 1 mM 3‐Aminotriazole (3AT). Note in (A) that the ^mCenp‐FR2687E mutant, but none of the other mCenp‐F mutants assayed in Fig 2B, interacts with full‐length mNup133. However, unlike WT mCenp‐F‐SID, the interaction of the R2687E mutant with full‐length mNup133 is abrogated by the addition of 1 mM 3‐Aminotriazole, indicating that the ^mCenp‐FR2687E mutant may interact with the full‐length mNup133 in a rather weak manner. In (B), note that the lexA‐mNup133‐CTD construct is transactivating when used in ‐LWH medium. Under conditions required to prevent this transactivation (i.e., addition of 1 mM 3‐Aminotriazole), the interaction of Nup133‐Cterm with its established C‐terminal partner, Nup107, is preserved, while no interaction was detected between Nup133‐Cterm and mCenp‐F‐SID, either WT or R2687E. This observation is consistent with our previous studies using human Nup133 constructs that indicated that the C‐terminal domain of Nup133 does not interact with Cenp‐F 13.
• C
  HeLa‐E cells transiently transfected with GFP‐mCenp‐F‐Ct2 R2687E were fixed 2 days after transfection and stained with an antibody directed against Elys that labels the NPCs and the GLFG bodies in interphase cells. The corresponding Western blot is presented in Appendix Fig S3. Scale bar, 10 μm. Note that consistent with the Y2H interaction observed with full‐length Nup133 [aa 1–1,155], GFP‐mCenp‐F‐Ct2 R2687E localizes in the GLFG bodies.