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Results: 10

1.
FIG. 9.

FIG. 9. From: Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification .

Notch-dependent T-cell lineage allocation in ex vivo OP9-DL1 coculture requires the N1-ICD binding domain of MTG16. Wild-type or Mtg16−/− LSK hematopoietic progenitors were transduced with empty MIG vector (A and B), MIG-myc-Mtg16 (C), or MIG-myc-Mtg16Δ2N (D) as shown. After coculture over OP9-DL1 monolayers, GFP-positive progeny cells were subjected to flow cytometry analysis with anti-CD4 and anti-CD8 antibodies.

Michael E. Engel, et al. Mol Cell Biol. 2010 April;30(7):1852-1863.
2.
FIG. 1.

FIG. 1. From: Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification .

Notch target gene expression is altered in Mtg16−/− LSK hematopoietic progenitors. LSK progenitors were purified from total bone marrow cells of Mtg16+/+ and Mtg16−/− mice. RNA was reverse transcribed and cDNA amplified by real-time PCR. Results are presented as fold change in gene expression from Mtg16−/− LSK cells relative to Mtg16+/+ LSK cells. Asterisks indicate P < 0.05.

Michael E. Engel, et al. Mol Cell Biol. 2010 April;30(7):1852-1863.
3.
FIG. 4.

FIG. 4. From: Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification .

MTG16 binds the intracellular domains of each Notch receptor. The intracellular domains of Notch1 through Notch4 were expressed alone or with myc-MTG16. Epitope tags were incorporated at the C termini for each N-ICD. N1-, N2-, and N3-ICDs incorporate Flag epitope tags, while N4-ICD has an HA epitope tag. N-ICDs were purified on protein G-Sepharose beads with anti-Flag M2 or anti-myc 9E10 antibodies as shown (IP). For N1- through N3-ICDs, coprecipitating MTG16 was identified by immunoblotting of anti-Flag immune complexes, while coprecipitating N4-ICD was identified by immunoblotting of anti-myc immune complexes. Expression of N-ICDs and MTG16 was confirmed in whole-cell lysates. Equal precipitations were confirmed by anti-Flag or anti-myc immunoblots of anti-Flag or anti-myc immune complexes, respectively. Equal transfections were confirmed by immunoblotting for GFP expressed from a cotransfected, constitutive GFP expression plasmid. Equal protein loadings were confirmed by immunoblotting for α-tubulin.

Michael E. Engel, et al. Mol Cell Biol. 2010 April;30(7):1852-1863.
4.
FIG. 5.

FIG. 5. From: Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification .

N1-ICD disrupts the interaction between CSL and MTG16. Myc-CSL and HA-MTG16 were expressed together in COS7L cells either without or with increasing amounts of N1-ICD-Flag. CSL complexes were purified on protein G Sepharose with anti-myc antibody 9E10 and then analyzed by immunoblot analysis for CSL, MTG16, and N1-ICD in anti-myc immune complexes by using anti-myc A-14, anti-HA Y-11, and anti-Flag M2 antibodies. MTG16, N1-ICD, and CSL expression were determined in whole-cell lysates with epitope tag-specific antibodies. Equivalent transfection and gel loading were confirmed by GFP and α-tubulin expression, respectively. In parallel, myc-CSL, HA-MTG16, and N1-ICD-Flag were expressed alone and subjected to anti-myc immune precipitation and immunoblot analysis as described above. As additional controls, N1-ICD-Flag was expressed together with either HA-MTG16 or myc-CSL followed by anti-myc IP and immunoblot analysis as shown.

Michael E. Engel, et al. Mol Cell Biol. 2010 April;30(7):1852-1863.
5.
FIG. 8.

FIG. 8. From: Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification .

N-ICDs bind the MTG16 N terminus in vitro. The intracellular domains of Notch1 (N1-ICD) and Notch3 (N3-ICD) were transcribed and translated in vitro in the presence of biotin-lysyl-tRNA in rabbit reticulocyte lysate. An aliquot of each reaction mixture (input) was separated by SDS-PAGE, transferred to nitrocellulose, and probed with the streptavidin-HRP conjugate. The remainder of each reaction mixture was incubated with bacterially expressed GST or GST-NHR1 bound to glutathione agarose beads as shown. Beads were washed with 1× PBS-T, and precipitated proteins were separated by SDS-PAGE. After transfer to nitrocellulose, biotinylated N1-ICD or N3-ICD coprecipitating with GST or GST-NHR1 was detected using streptavidin-HRP and chemiluminescence (ppt). Aliquots of purified GST and GST-NHR1 used for in vitro binding assays are shown (CBB).

Michael E. Engel, et al. Mol Cell Biol. 2010 April;30(7):1852-1863.
6.
FIG. 10.

FIG. 10. From: Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification .

Model for MTG16-mediated regulation of the canonical Notch transcription complex. In the unstimulated state, MTG16, represented as an antiparallel tetramer, serves as a scaffold for corepressors (Corep's) and HDACs to repress Notch target gene expression. 1, Activation of Notch signaling liberates the N-ICD from the plasma membrane, allowing its transit to the nucleus; 2, interaction with MTG16 and CSL induces conformational changes that destabilize the repressor complex, promoting release of MTG16, other corepressors and HDACs; 3, binding of Mastermind (MAML) and general transcriptional coactivators/histone acetyltransferases (Co-act) promotes recruitment of the basal transcription machinery and endorses Notch target gene expression.

Michael E. Engel, et al. Mol Cell Biol. 2010 April;30(7):1852-1863.
7.
FIG. 3.

FIG. 3. From: Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification .

N1-ICD interacts with MTG16, but not NCoR, SIN3A, or HDAC3. (A) Flag-tagged N1-ICD was transiently expressed in COS7L cells alone, with myc-CSL or myc-MTG16. Coprecipitating CSL and MTG16 were identified in anti-Flag (α-Flag) immune complexes by anti-myc immunoblot analysis with A-14. Equivalent N1-ICD-Flag precipitation was confirmed by anti-Flag immunoblot analysis of Flag immune complexes. (B) Flag epitope-tagged NCoR, SIN3A, or HDAC3 was transiently expressed in COS7L cells, alone or with N1-ICD. Anti-Flag immune complexes were probed with bTan20 or anti-Flag M2. N1-ICD expression was confirmed in whole-cell lysates (WCL). GFP and α-tubulin expression were used for transfection and loading controls, respectively. (C) Whole-cell lysates were prepared from Jurkat T-cell leukemia cells and subjected to immune precipitation with nonimmune goat or mouse sera (NGS and NMS, respectively) or with α-MTG16 antibody G-20 or bTan20. Immune complexes were separated by SDS-PAGE and subjected to immunoblot analysis with anti-MTG16 (α-MTG16) polyclonal antibody AJ/R (see Materials and Methods) or bTan20 as indicated. N1-ICD and MTG16 expression were confirmed in whole-cell lysates and equal loading confirmed with anti-α-tubulin immunoblotting.

Michael E. Engel, et al. Mol Cell Biol. 2010 April;30(7):1852-1863.
8.
FIG. 2.

FIG. 2. From: Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification .

MTG family proteins bind CSL. (A) Myc epitope-tagged CSL was expressed alone or with HA epitope-tagged versions of MTG16, MTGR1, or RUNX1-MTG8 in COS7L cells and subjected to immune precipitation (IP) with anti-myc (α-myc) antibody 9E10. Coprecipitating MTG proteins were identified in anti-myc immune complexes (IC) by immunoblot analysis with anti-HA (α-HA) antibody Y-11. Expression of each protein was determined by immunoblotting with epitope tag-specific antibodies. Equivalent loading was confirmed by α-tubulin-specific immunoblotting. (B) Myc epitope-tagged CSL was expressed alone or with Gal epitope-tagged MTG16 constructs expressing each NHR (NHR1 to -4) with its N-terminal and C-terminal flanking structures. Graphical representations of each MTG16 construct are shown relative to full-length MTG16. NHR domains are numbered 1 to 4, and the PST regions are indicated. CSL was identified by an A-14 anti-myc immunoblot of α-Gal immune complexes. Expression of CSL and MTG16 derivatives were confirmed by epitope tag-specific immunoblotting.

Michael E. Engel, et al. Mol Cell Biol. 2010 April;30(7):1852-1863.
9.
FIG. 6.

FIG. 6. From: Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification .

MTG16-CSL cosedimentation is abolished by N1-ICD expression. (A to D) Myc-CSL, HA-MTG16, and N1-ICD-Flag were expressed in COS7L cells individually (A) or in the combinations shown (B, C, and D). Whole-cell extracts were prepared under nondenaturing conditions and fractionated through preprepared 10 to 40% (wt/wt) gradients of buffered sucrose. Fractions were drawn through nitrocellulose membranes by using a vacuum manifold and slot blot apparatus to allow protein binding. Protein binding was confirmed with Ponceau S staining. Nitrocellulose membranes were subjected to immunoblot analysis using epitope tag-specific antibodies and fluorescence detection with a Li-Cor Odyssey system. Vertical bars denote distinct regions of the membrane reorganized into a linear array for presentation purposes. Fraction numbers are indicated along the top of panel A. Sedimentation controls were fractionated through a gradient prepared and processed in parallel. (E) Aliquots of whole-cell extracts were analyzed in parallel by SDS-PAGE and immunoblotting to confirm specific detection of each protein. MW, molecular mass.

Michael E. Engel, et al. Mol Cell Biol. 2010 April;30(7):1852-1863.
10.
FIG. 7.

FIG. 7. From: Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification .

N1-ICD binding maps to the N terminus of MTG16, distinct from conserved NHR domains. (A) N1-ICD-Flag was expressed alone, with myc-MTG16 or with myc-tagged fragments of MTG16 that are serially deleted from the N and C termini (A and C, respectively). Extracts were subjected to immune precipitation with anti-Flag antibody M2. N1-ICD-bound MTG16 or its truncated derivatives were identified in anti-Flag immune complexes by immunoblot analysis with anti-myc antibody A-14. N1-ICD precipitation was confirmed by anti-Flag immunoblotting of anti-Flag immune complexes. Expression of N1-ICD, MTG16, and truncated derivatives of MTG16 were confirmed by immunoblot analysis of whole-cell lysates by using Flag- and myc-specific antibodies. (B and D) Graphical representation of MTG16 and N-terminal and C-terminal deletion mutants. An open circle represents the N-terminal myc tag in each construct. The NHR domains are labeled 1 through 4. Proline-serine-threonine (PST) regions 1, 2, and 3 are indicated. Amino acid boundaries for each MTG16 fragment are shown in parentheses. (E) The primary structure of the N1-ICD binding region on MTG16 is indicated. WT, wild type.

Michael E. Engel, et al. Mol Cell Biol. 2010 April;30(7):1852-1863.

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