1. Volume 13, Issue 4, Pages 587-597 (February 2004)
Menin Associates with a Trithorax Family Histone Methyltransferase Complex and with the Hoxc8 Locus 
Christina M. Hughes, Orit Rozenblatt-Rosen, Thomas A. Milne, Terry D. Copeland, Stuart S. Levine, Jeffrey C. Lee, D. Neil Hayes, Kalai Selvi Shanmugam, Arindam Bhattacharjee, Christine A. Biondi, Graham F. Kay, Nicholas K. Hayward, Jay L. Hess, Matthew Meyerson 
Molecular Cell 
Volume 13, Issue 4, Pages (February 2004)
DOI: /S (04)
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2. Figure 1
Anti-Menin Antibody Characterization and Identification of Interacting Proteins
(A) Lysates of 293T cells were immunopre-cipitated with anti-menin 342 antibody or peptide-blocked control. Anti-menin immuno-precipitate, peptide-blocked control immunoprecipitate, and 50 μg whole cell lysate were resolved by 8% SDS-PAGE and immunoblotted with anti-menin antibody 342.
(B) Immunoprecipitation from metabolically labeled HeLa cells using anti-menin (342) antibody and peptide-blocked control, resolved by 9% SDS-PAGE. Numbered bands were identified by a repeat immunoprecipitation subjected to Coomassie blue staining and mass spectrometric analysis: 1, Rpb2; 2, MLL2; 3, Ash2L; 4, menin; 5, Rbbp5; and 6, WDR5. The asterisk denotes a band that was not identified as it comigrated with immunoglobulin heavy chain.
Molecular Cell  , DOI: ( /S (04) )
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3. Figure 2 Menin Interacts Specifically with Rbbp5, Ash2L, and MLL2
(A) Anti-menin (lane 3) and anti-Rbbp5 (lane 5) immunoprecipitates from 293T cell lysates were fractionated and immunoblotted with anti-menin 342 antibody (top panel) or anti-Rbbp5 antibody (bottom panel). Cell lysate was loaded in lane 1. Negative control immunoprecipitations were performed with normal rabbit IgG (lane 2) and peptide-blocked anti-menin 342 and anti-Rbbp5 antibodies (lanes 4 and 6).
(B) Anti-menin (lane 3) and anti-Ash2L 867 (lane 5) immunoprecipitates were immunoblotted with anti-menin 342 antibody (top panel) or anti-Ash2L C19 antibody (bottom panel). Normal rabbit IgG (lane 2) and peptide-blocked anti-menin 342 and anti-Ash2L 867 (lanes 4 and 6) were used as controls.
(C) Immunoprecipitations using anti-menin (lane 3) and anti-Flag (lane 5) antibodies, from 293T cell lysates transfected with Flag-MLL2 and menin, were immunoblotted with anti-menin (top panel) or anti-Flag (bottom panel) antibodies. Lysate from transfected cells was loaded in lane 1 and mock-transfected cells in lane 7. Normal mouse IgG (lane 2) and peptide-blocked anti-menin 342 antibody (lane 4) were used for control immunoprecipitations.
Molecular Cell  , DOI: ( /S (04) )
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4. Figure 3
The Menin HMTase Complex Is Comprised of Homologs of Members of the Yeast Set1 Complex and Three Human Set1-like Complexes, ASCOM, a Complex that Associates with HCF-1, and the MLL1 Complex
SET, SET domain; post-SET, cysteine rich domain associated with the SET domain; FY, phenylalanine/tyrosine-rich domain; SPRY, domain in SPla and the RYanodine receptor; PHD, PHD zinc finger motif; WD40, WD repeat domain; and RIIa, protein kinase A regulatory subunit dimerization domain. Figure adapted with permission from Wysocka et al. (2003).
Molecular Cell  , DOI: ( /S (04) )
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5. Figure 4 The Menin HMTase Complex Methylates Histone 3 Lysine 4
(A) Menin or peptide-blocked control immunoprecipitates were incubated with histone H3 (lanes 1 and 2) or core histones (lanes 3 and 4) and the methyl donor 3H-SAM. Samples were resolved on 15% SDS-PAGE, stained with Coomassie blue (top panel), amplified, dried, and flourographed (bottom panel). Histone subunits are identified on the right.
(B) Menin WT (lanes 1 and 2) and KO (lanes 3 and 4) cells were immunoprecipitated with anti-menin 342 antibody, and HMTase assays were performed as described above.
(C and D) Scintillation counting of sequential Edman degradation products of histone H3, labeled by incubation with 3H-SAM and immunoprecipitates of menin WT (C) and KO (D) cells. Note the different scales of panels (C) and (D). The corresponding amino acid residues are indicated below each graph.
Molecular Cell  , DOI: ( /S (04) )
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6. Figure 5
HMTase Activity Is Not Associated with a Subset of Patient-Derived Menin Point Mutants
Men1 KO cells were infected with pBABE retroviruses containing either vector alone (KO + pBABE), WT menin (KO + pBABE menin), or one of seven menin point mutants identified from MEN1 patients: P12L, L22R, H139D, A242V, A309P, T344R, or W436R. Lysates from each transfected cell type, along with KO cells, Men1+/+ matched WT MEFs, and 293T cells were immunoprecipitated and immunoblotted for menin (bottom panel). Menin immunoprecipitates were subjected to HMTase assay, analyzed by 15% SDS-PAGE, and fluorographed (top panel).
Molecular Cell  , DOI: ( /S (04) )
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7. Figure 6
Menin Interacts with the Ser 5 Phosphorylated Form of RNA Polymerase II Large Subunit
293T cell lysates were immunoprecipitated with anti-menin 342 antibody (middle lane) or peptide-blocked control (right lane), and immunoblotted for Rpb1 with Ser 2 phosphorylated CTD (top panel, H5 immunoblot), Ser 5 phosphorylated CTD (2nd panel, H14 immunoblot), or unphosphorylated CTD (3rd panel, 8WG16 immunoblot), and for menin (bottom panel).
Molecular Cell  , DOI: ( /S (04) )
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8. Figure 7 Menin Regulates Hox Gene Expression in MEFs
(A) Expression of Hoxc8 and Hoxc6 was measured using quantitative real-time RT-PCR in Men1+/+ or −/− MEFs, as well as in Men1 KO MEFs that had either been infected with retroviral vector alone (KO + vec) or retroviral vector plus menin (KO + menin).
(B) Positions of the Hoxc8 probes (5′E, c8P2, and c8P3) used for chromatin immunoprecipitations. Base positions refer to accession number M35603 (Awgulewitsch et al., 1990). The arrow indicates the transcriptional start site and the open box indicates the beginning of the coding sequence.
(C) Menin binds to the Hoxc8 promoter. Chromatin immunoprecipitations were done for menin or a control using anti-menin 342 and anti-rabbit IgG, and gene binding was tested using real-time PCR for three Hoxc8 probes and a Gapdh probe.
Molecular Cell  , DOI: ( /S (04) )
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