Figure 2

Domain mapping of the Notch1 intracellular domain-Snail complex. (A) Schematic of Notch1 intracellular domain (NICD) and deletion mutants. The ability of each fragment to bind Snail is indicated on the right. (B) 293T cells were cotransfected with Flag-Snail and Myc-NICD deletion mutants. After transfection, cell lysates were immunoprecipitated with anti-Flag antibody and assessed by immunoblot analysis. The NICD ankyrin/cdc10 repeat (ANK) domain ΔANK mutant did not bind the Snail protein. NLS, nuclear localization signal. (C) Schematic of Snail and deletion mutants. The ability of each fragment to bind NICD is indicated on the right. (D) 293T cells were cotransfected with Myc-NICD and Flag-Snail deletion mutants. After transfection, cell lysates were immunoprecipitated with anti-Myc antibody and assessed by immunoblot analysis. The Snail zinc finger (ZF) ΔZF mutant did not bind the NICD protein. (E) Glutathione S-transferase (GST)-Snail wild-type (WT), ΔZF and ZF mutants were incubated with cell lysates of Huh7 cells that overexpressed Myc-NICD. The bound proteins were eluted by boiling the cells in SDS sample buffer for 10 minutes and then were analyzed by immunoblotting with anti-Myc and anti-GST antibodies.