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Fig. 2. Zfp64 associates with NICD. (A) Yeast-two-hybrid assay. The PEST-containing region of Notch1 interacts with Zfp64 and its C-terminal region (Zfp-C'), and also with the C-terminal region of the other Zfp64 isoforms (Zfp-C); this was observed by formation of blue colony. Zfp-N, which is the N-terminal region of all the Zfp64 isoforms, showed no interaction with the PEST-containing region. The mock vector and lamin C (Lam) showed no interaction with Zfp64. (B) Translation of the genes fused with a GAL4 activation domain in each yeast transformants was confirmed by Western blot analysis using anti-HA antibody. Translation of the genes fused with a GAL4 DNA-binding domain was confirmed using anti-Myc antibody. (C) Zfp64 associates with the PEST-containing region of Notch1. HA-tagged Zfp64 and Myc-tagged PEST were co-synthesized in vitro using a rabbit reticulocyte lysate system. Immunoprecipitation of the PEST-containing region using anti-Myc antibody revealed co-precipitation of Zfp64. (D) Zfp64 associates with NICD in cells. HA-tagged Zfp64 and N-terminal FLAG-tagged NICD or NICD-p were co-transfected into HEK293 cells. Protein extraction and immunoprecipitation were performed in three different lysis buffers: RIPA, RIPA with EDTA, and RIPA with zinc acetate. Immunoprecipitation of NICD using anti-Flag antibody revealed the association of Zfp64 and NICD in RIPA with zinc acetate. A small amount of Zfp64 co-precipitant was detected in RIPA and the least amount of co-precipitant was detected in RIPA with EDTA. Zfp64 also co-precipitated with NICD-p in RIPA with zinc acetate. (E) Endogenous Zfp64 co-precipitated with adenovirally delivered NICD. U2OS cells were transfected with AdFlg-NICD-V5 and immunoprecipitation with anti-FLAG antibody was performed.
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