The N-terminal domain of hNup133 is largely dispensable for NPC assembly. (A) Control HeLa cells (WT) or cell lines stably expressing GFP-hNup133_CTD or GFP_3x-mNup133 were transfected with a scramble siRNA or with a hNup133 siRNA that does not target hNup133_CTD and only mildly impacts mNup133 expression. After 3 d, whole-cell extracts were analyzed by Western blot using anti-hNup133 (which also recognizes mouse Nup133, albeit less efficiently than human Nup133) or anti-hNup107 antibodies. Anti–γ-tubulin was used as loading control. To allow quantifications, decreasing amounts of the reference sample were loaded (0.2-, 0.5-, and 1-fold equivalent). (B) Cells transfected with siRNAs as in A were fixed after 3 d and processed for immunofluorescence using anti-Nup107 and mAb414 antibodies. Bar, 20 µm.