Figure 5.

Depletion of Nudel downregulates the levels of the WRC. (A) Nudel was removed from the tetrameric subcomplexes as well as mature WRC. HEK293T cells transfected with the indicated amount of plasmids were lysed and subjected to co-IP with anti-Flag resin. (B) Endogenous Nudel and WRC did not coexist in the same fraction after sucrose gradient ultracentrifugation of HeLa cell lysate. (C) Nudel depletion attenuated the levels of both endogenous WRC subunits and mature WRC. HEK293T cells were transfected twice with the control (pTER-Luci) or Nudel RNAi (pTER-Nudi) plasmid, respectively, and harvested at 84 h. Lysates from equal number of cells (lanes 2 and 3) were subjected to co-IP with anti-Abi1 antibody (lanes 5 and 6), whereas lysate from untransfected cells (lane 1) was immunoprecipitated with normal IgG to serve as a control (lane 4). (D) The quantification results for the relative protein levels in C. The data were averaged from three independent experiments. *, **, and *** represent P ≤ 0.05, 0.01, and 0.001, respectively, in Student's t-tests. (E) The levels of mature WRC were reduced in Nudel-depleted cells. HeLa cells were transfected twice with pTER-Luci or pTER-Nudi, respectively, and harvested at 84 h. After the sucrose gradient ultracentrifugation, the indicated fractions of control (L) and Nudel-depleted (N) lysates were subjected to SDS-PAGE. Equal loading of matched factions was confirmed by the Ponceacu S staining prior to immunoblotting.