FIG. 5.

The Notch TAD physically interacts with the LEF-1 HMG domain. (A) LEF-1 activates Gal4-NICDΔRA. Neuro-2A cells were transfected with a Gal4 responsive reporter and either the DNA binding domain of Gal4 (Gal4) or a Gal4 fused to the TAD of Notch (Gal4-NICDΔRA) in the presence (gray bars) or absence (black bars) of LEF-1 as indicated. Results are shown as fold induction relative to the reporter plus the Gal4 DNA binding domain. (B) LEF-1 interacts with the Notch TAD in vitro. 293T cells were transfected with plasmids expressing HA-LEF-1 and FLAG-CSL, extracts were incubated with GST, GST-NICDΔTAD, or GST-NICDΔRA, and bound proteins were analyzed by Western analysis using anti-HA antibodies (top panel) or anti-FLAG antibodies (lower panel). Samples of untreated cell extracts were loaded in each of the far left lanes, corresponding to 1/350 of the input analyzed for HA-LEF-1 (top) and 1/5 of the input analyzed for FLAG-CSL (bottom). (C) LEF interacts with the Notch TAD and ALY with comparable affinities. 293T cells were transfected with HA-LEF, extracts were incubated with GST, GST-NICDΔRA or GST-ALY as indicated, and bound proteins were analyzed by Western analysis with an anti-HA antibody. Lane 1 contains untreated extract corresponding to 1/300 of the input. (D) Schematic diagram of the LEF-1 fragments used to map the interaction with NICD. Amino acid positions and several functionally defined domains are indicated. βCID, β-catenin interaction domain; CAD, context-dependent activation domain; HMG, HMG domain. (E) The LEF-1 HMG box mediates interactions with the Notch TAD. The indicated radiolabeled LEF-1 fragments were generated by in vitro transcription and translation (lanes 1, 4, 7, 10, and 13) and analyzed for binding to GST (lanes 2, 5, 8, 11, and 14) and GST-NICDΔRA (lanes 3, 6. 9, 12, and 15). Untreated samples represent 1/100 of the input used for each binding analysis. Positions of molecular mass standards (in kilodaltons) are shown at the left.