Fig. 2.
Fig. 2. TEL and TEL-AML1 interact with N-CoR in vivo. / (A) Mammalian 2 hybrid analysis of interaction between the entire N-CoR, SMRT, or mSin3A (as a positive control) fused to the GAL4(DBD) and VP16-activation domain tagged TEL or TEL-AML1. In agreement with the in vitro co-immunoprecipitation results, full-length AML1 fused to the VP16-activation domain failed to interact with GAL4(DBD)-N-CoR. No significant interaction was also detected in this assay between SMRT and TEL or AML1. Interactions between VP16-TEL and GAL4(DBD)-mSin3A are shown as a positive control. Interactions between GAL4(DBD)-N-CoR or GAL4(DBD)-SMRT and VP16 tagged RARα in the absence or presence of all-trans-retinoic acid (RA) are used as additional positive and negative controls, respectively. Co-transfection of an empty GAL4(DBD) vector (pGALO) either with VP16-TEL or VP16-AML1 did not result in activation of the luciferase gene expression (not shown). (B) Co-immunoprecipitation of endogenous human (h) N-CoR and TEL-AML1 (lanes 5-7) or TEL (lane 8) from 293T cells transfected with their respective expression vectors. Lanes 6 and 7 show decreasing levels of the co-immunoprecipitated TEL-AML1 protein with increasing amounts of the N-CoR antigenic peptide in the reaction. Input (20%) is shown in lanes 1-3. Lane 3 corresponds to protein extract derived from untransfected 293T cells. Size markers in kd are indicated on the left of the panel. (C) Antibodies against human N-CoR co-immunoprecipitate the endogenous TEL-AML1 protein from REH cells with t(12;21). Proteins were co-immunoprecipitated from whole cell extracts using polyclonal antibodies specific against human N-CoR (lane 2), murine N-CoR (lane 5), mSin3A (lane 6), TEL (lane 7), and AML1 (lane 8), as indicated. Immunoprecipitated material was resolved by SDS-PAGE and Western blotting using anti-TEL antibody. Lanes 3 and 4 correspond to co-immunoprecipitation carried out in the presence of 0.6- and 1.4-μg N-CoR antigenic peptide. Lane 1 represents 20% of the input for the co-immunoprecipitation reaction. Size markers in kd are indicated on the left of the panel.

TEL and TEL-AML1 interact with N-CoR in vivo.

(A) Mammalian 2 hybrid analysis of interaction between the entire N-CoR, SMRT, or mSin3A (as a positive control) fused to the GAL4(DBD) and VP16-activation domain tagged TEL or TEL-AML1. In agreement with the in vitro co-immunoprecipitation results, full-length AML1 fused to the VP16-activation domain failed to interact with GAL4(DBD)-N-CoR. No significant interaction was also detected in this assay between SMRT and TEL or AML1. Interactions between VP16-TEL and GAL4(DBD)-mSin3A are shown as a positive control. Interactions between GAL4(DBD)-N-CoR or GAL4(DBD)-SMRT and VP16 tagged RARα in the absence or presence of all-trans-retinoic acid (RA) are used as additional positive and negative controls, respectively. Co-transfection of an empty GAL4(DBD) vector (pGALO) either with VP16-TEL or VP16-AML1 did not result in activation of the luciferase gene expression (not shown). (B) Co-immunoprecipitation of endogenous human (h) N-CoR and TEL-AML1 (lanes 5-7) or TEL (lane 8) from 293T cells transfected with their respective expression vectors. Lanes 6 and 7 show decreasing levels of the co-immunoprecipitated TEL-AML1 protein with increasing amounts of the N-CoR antigenic peptide in the reaction. Input (20%) is shown in lanes 1-3. Lane 3 corresponds to protein extract derived from untransfected 293T cells. Size markers in kd are indicated on the left of the panel. (C) Antibodies against human N-CoR co-immunoprecipitate the endogenous TEL-AML1 protein from REH cells with t(12;21). Proteins were co-immunoprecipitated from whole cell extracts using polyclonal antibodies specific against human N-CoR (lane 2), murine N-CoR (lane 5), mSin3A (lane 6), TEL (lane 7), and AML1 (lane 8), as indicated. Immunoprecipitated material was resolved by SDS-PAGE and Western blotting using anti-TEL antibody. Lanes 3 and 4 correspond to co-immunoprecipitation carried out in the presence of 0.6- and 1.4-μg N-CoR antigenic peptide. Lane 1 represents 20% of the input for the co-immunoprecipitation reaction. Size markers in kd are indicated on the left of the panel.

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