NCL binds to the C-terminal domain of CLTA.

(A) Screening of host proteins that interacted with the NCL protein. SDS-PAGE analysis of immunoprecipitation-purified host factors of RK-13 cells that interact with the NCL protein was performed with NCL mAb. The bands were identified by mass spectrometry. In addition, M is a protein marker and the negative control was cell lysates immunoprecipitation-purified with mouse IgG. (B) Validation of the interaction between CLTA and NCL with the IP assay. Proteins in the lysates of RK-13 cells were immunoprecipitated with anti-CLTA mAb, anti-AP2 mAb, or mouse IgG, and then immunoblotted with anti-NCL mAb. β-actin was employed as an internal control. (C) Schematic illustration of the full-length and truncated forms of GST-CLTA for the GST pull-down assay. The numbers indicate aa positions. (D) GST-CLTA proteins were purified on glutathione-sepharose beads and incubated in RK-13 cells lysates containing the NCL protein. After extensive washing, the binding of NCL was determined by western blot analysis with anti-NCL mAb. The GST protein acted as a negative control.