Fig. 4.
Fig. 4. Mapping of the intramolecular interaction domains. / (A) Schematic representation of the various MLL deletion mutants tagged with HA at their C-termini and various GAL4-MLL C-terminal fragments tagged with His at their N-termini. The known motifs and domains of the AT hook (171-309), NTS-1 (244-314), SNL-1 (388-432), NTS-3 (727-762), SNL-2 (1050-1089), the MT domain (1136-1203), NIID (1253-2254), the PHD fingers (1418-1628), the bromo domain (1633-1769), the Zn fingers (Zn: 1870-1977), the processing sites (D2666 and D 2718), the transactivation domain (denoted as AD, 2829-2883), CIID (3607-3742), and the SET domain (3818-3969) are shown.4046 Interaction between N- and C-terminal fragments is also shown on the right as present (+) or absent (–). (B) Determination of N-terminal edge of the N-terminal intramolecular interaction domain (NIID). BOSC23 cells were transfected with the pM vector or one of the pM MLL(H) vectors expressing N-terminal deletion mutants of MLL fused with GAL4 at their N-termini and tagged with HA at their C-termini. The cells were harvested and lysed in lysis buffer 48 hours after transfection. The GAL4-MLL proteins were immunoprecipitated with the anti-GAL4 antibody. The whole cell extracts (WCE, lanes 1-6) and the immunoprecipitates (IP:GAL4, lanes 7-12) were analyzed by immunoblot with anti-HA antibody 3F10. Molecular weight markers are on the left. (C, D) Determination of the C-terminal edge of the N-terminal intramolecular domain. BOSC23 cells were transfected with pLNCX vectors expressing C-terminal deletion mutants of MLL and pLNCX vector expressing GAL4-MLL 2719/3968 tagged with His at the N-termini. The MLL proteins were immunoprecipitated with anti-MLL N-terminal antibody (mmN3). WCE and the precipitates were separated in denaturing gels and immunoblotted with anti-MLL N-terminal antibody (mmN3) (WCE only; C) or anti-His antibody (D-8; D). (E) Determination of the C-terminal edge of the C-terminal intramolecular interaction domain. BOSC23 cells were transfected with the pLNCX vectors expressing C-terminal deletion mutants of MLL tagged with HA at their C-termini. The MLL proteins were immunoprecipitated with the anti-MLL N-terminal antibody (mmN3) antibody. WCE (lanes 1-6) and the immunoprecipitates (lanes 7-12) were immunoblotted with the anti-HA antibody 3F10. Panels F and G show the determination of the N-terminal edge of the C-terminal intramolecular interaction domain. BOSC23 cells were transfected with pLNCX vectors expressing MLL1/2666 and pLNCX vectors expressing N-terminal deletion mutants of GAL4-MLL 2719/3968 tagged with His at the N-termini. The MLL proteins were immunoprecipitated with anti-MLL N-terminal antibody (mmN3). The whole cell extracts and the precipitates were separated in denaturing gels and immunoblotted with anti-MLL N-terminal antibody (mmN3) (WCE only; F) or anti-His antibody (D-8; G).

Mapping of the intramolecular interaction domains.

(A) Schematic representation of the various MLL deletion mutants tagged with HA at their C-termini and various GAL4-MLL C-terminal fragments tagged with His at their N-termini. The known motifs and domains of the AT hook (171-309), NTS-1 (244-314), SNL-1 (388-432), NTS-3 (727-762), SNL-2 (1050-1089), the MT domain (1136-1203), NIID (1253-2254), the PHD fingers (1418-1628), the bromo domain (1633-1769), the Zn fingers (Zn: 1870-1977), the processing sites (D2666 and D 2718), the transactivation domain (denoted as AD, 2829-2883), CIID (3607-3742), and the SET domain (3818-3969) are shown.40 46 Interaction between N- and C-terminal fragments is also shown on the right as present (+) or absent (–). (B) Determination of N-terminal edge of the N-terminal intramolecular interaction domain (NIID). BOSC23 cells were transfected with the pM vector or one of the pM MLL(H) vectors expressing N-terminal deletion mutants of MLL fused with GAL4 at their N-termini and tagged with HA at their C-termini. The cells were harvested and lysed in lysis buffer 48 hours after transfection. The GAL4-MLL proteins were immunoprecipitated with the anti-GAL4 antibody. The whole cell extracts (WCE, lanes 1-6) and the immunoprecipitates (IP:GAL4, lanes 7-12) were analyzed by immunoblot with anti-HA antibody 3F10. Molecular weight markers are on the left. (C, D) Determination of the C-terminal edge of the N-terminal intramolecular domain. BOSC23 cells were transfected with pLNCX vectors expressing C-terminal deletion mutants of MLL and pLNCX vector expressing GAL4-MLL 2719/3968 tagged with His at the N-termini. The MLL proteins were immunoprecipitated with anti-MLL N-terminal antibody (mmN3). WCE and the precipitates were separated in denaturing gels and immunoblotted with anti-MLL N-terminal antibody (mmN3) (WCE only; C) or anti-His antibody (D-8; D). (E) Determination of the C-terminal edge of the C-terminal intramolecular interaction domain. BOSC23 cells were transfected with the pLNCX vectors expressing C-terminal deletion mutants of MLL tagged with HA at their C-termini. The MLL proteins were immunoprecipitated with the anti-MLL N-terminal antibody (mmN3) antibody. WCE (lanes 1-6) and the immunoprecipitates (lanes 7-12) were immunoblotted with the anti-HA antibody 3F10. Panels F and G show the determination of the N-terminal edge of the C-terminal intramolecular interaction domain. BOSC23 cells were transfected with pLNCX vectors expressing MLL1/2666 and pLNCX vectors expressing N-terminal deletion mutants of GAL4-MLL 2719/3968 tagged with His at the N-termini. The MLL proteins were immunoprecipitated with anti-MLL N-terminal antibody (mmN3). The whole cell extracts and the precipitates were separated in denaturing gels and immunoblotted with anti-MLL N-terminal antibody (mmN3) (WCE only; F) or anti-His antibody (D-8; G).

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