Identification and nuclear localization of the hMam-1 protein. (a) Identification of the hMam-1 protein. 293T cells were transfected with an expression vector for hMam-1 N-HA or the empty control vector (pEF-BOS). Whole-cell extracts prepared from these cells were analyzed by immunoblotting using an anti-HA antibody. (b) Nuclear localization of the hMam-1 protein. 293T cells transfected with the expression vector for hMam-1 N-HA or the empty control vector (pEF-BOS) were stained with the anti-HA antibody and a fluorescein isothiocyanate-conjugated secondary antibody. Phase-contrast, fluorescent, and merged views are shown.

Primary structure of Mam proteins. (a) Primary sequences of DMam (GenBank accession number X54251) and hMam-1 (D83785). Sequences were aligned using the ClustalW algorithm. Identical amino acids are in shaded boxes. Similar amino acids are in open boxes. The basic and acidic regions are in labeled boxes. The residues converted in the deletion mutants of hMam-1 are indicated by arrowheads. The overall identity of hMam-1 and DMam is 22%. Identity over the most highly conserved basic region is 38%. (b) Arrangement of basic and acidic regions of the DMam and hMam-1 proteins.

Effects of full-length and truncated forms of hMam-1 on transactivation of the HES-5 promoter. NIH 3T3 cells were transfected with expression vectors for Notch and Mam or their empty counterparts as controls. The transfection also contains the pHES-5 luciferase reporter and an internal control for transfection. Vertical axes represent normalized luciferase activity relative to the mean activity of empty vector-transfected cells. Error bars indicate standard deviations (n = 3). (a) hMam-1 and Notch1IC synergistically activate the target promoter. (b) hMam-1 truncated at proline residue 103 depresses the activity induced by Notch1IC.

DMam function in cell culture. (a) Physical association of DMam with DNotchIC and Su(H). S2 cell extracts were immunoprecipitated with an anti-c-Myc antibody. The precipitates were resolved on an SDS gel, transferred to a membrane, and stained sequentially with anti-c-Myc, anti-Notch, and anti-DMam antibodies. Lane 2 shows that DMam is expressed endogenously in S2 cells. (b) DMam augments the activation of an E(spl) mγ reporter. The vertical axis represents normalized relative luciferase activity. The cotransfection of DMam with DNotch ICN (DNotchIC) and Su(H) leads to a doubling of reporter activity. Five experiments were performed, and all data was obtained in triplicate. Error bars indicate standard errors of the means.

Formation of the Mam-Notch-CSL complex on its target promoter elements. (a) Extracts of transfected 293T cells exhibit activities that bind to the RBP-J element of the HES-1 promoter. Cells were transfected with the expression vectors for the indicated proteins. NS designates a nonspecific complex, open arrowheads mark RBP-J-specific bands, the closed arrowhead marks the RBP-J–Notch1IC-specific band, and stars mark hMam-1–RBP-J–Notch1IC-specific bands. The closed star marks the hMam-1–RBP-J–Notch1IC band that could be supershifted by the anti-Notch1 and anti-HA antibodies (see panel b). (b) Effects of antibodies on binding activities. Complexes formed in the presence of RBP-J, RBP-J–Notch1IC, or hMam-1–RBP-J–Notch1IC were tested for supershifts after incubation with the indicated antibodies. Anti-CD44 was included as a control. NS, arrowheads, and stars are as described for panel a. (c) hMam-1 and its truncations containing the basic charge cluster can associate with RBP-J and Notch1IC within ternary complexes. The hMam-1 truncations, characterized below, were tested for the ability to form the ternary complex that is observed with full-length hMam-1. 293T cells were transfected with the indicated combinations of the expression vectors, and their extracts were analyzed for binding to the RBP-J element. The binding complexes migrate at a rate proportional to the length of the truncated form of hMam-1. Stars are as described for panel a. (d) Expression of the truncated forms of the hMam-1 protein. Extracts of 293T cells transfected with the vectors for indicated proteins were blotted and stained with anti-HA antibody. (e) Expression of hMam-1 enhances the physical association of RBP-J with Notch1IC. 293T cell extracts transfected with indicated vectors were immunoprecipitated by anti-Notch1 antibody. The precipitates were separated on an SDS gel, blotted onto a membrane, and then stained sequentially with anti-Notch1, anti-RBP-J, and anti-HA antibodies. The hMam-1 N-HA Pro103stop protein was too small to be resolved on this gel.