Figure 2.

The impact of the Nudel-Sra1 interaction. (A) The effect of Nap1, Abi1, and WAVE2 on the Nudel-Sra1 interaction. HA-tagged WRC subunits were overexpressed to similar levels in the indicated combinations in HEK293T cells, together with Flag-Nudel. The amount of each expression plasmid used is listed. The cell lysates were subjected to co-IP with anti-Flag resin. (B) The effect of Nudel on associations of Sra1, Nap1, and WAVE2 with Abi1. HA-tagged proteins were overexpressed to similar levels as indicated, together with Flag-Abi1, in HEK293T cells. The cell lysates were subjected to co-IP with anti-Flag resin. (C) The levels of Sra1, Nap1, and Abi1 increased when Nudel was coexpressed. The indicated proteins were overexpressed with the indicated amount of each plasmid. GFP was used as a control for transfection efficiency. (D) The quantification results for the relative protein levels in lanes 5 and 6 of C. The protein levels were normalized to those of GFP. The values in lane 6 are then presented in arbitrary units relative to corresponding ones in lane 5. *, ** and *** represent P ≤ 0.05, 0.01 and 0.001, respectively, in Student's t-tests. (E) Nudel increased the stability of the S-N-A complex. HEK293T cells were transfected as indicated in the lanes 5 and 6 of C, except that Flag-luciferase was expressed in the control cells. The cells were treated with 200 μg/ml of Chx at 36 h post transfection for the indicated times, followed by immunoblotting. (F) The quantification results for the relative protein levels of HA-Abi1 in E. The protein levels were normalized to those of GFP and presented relative to the values at 0 min.