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. Author manuscript; available in PMC: 2009 Jul 8.
Published in final edited form as: Cancer Cell. 2008 Jul 8;14(1):36–46. doi: 10.1016/j.ccr.2008.05.003

Figure 1. LEDGF and NUP98-LEDGF associate with the MLL/menin histone methyltransferase complex.

Figure 1

A. Silver stained image of an SDS-PAGE analysis shows presence of LEDGF in the purified MLL-ENL/menin immunoprecipitate. FLAG-tagged MLL-ENL [(f)ME] and HA-tagged menin [menin(H)] were transiently expressed in large scale culture of 293T cells. A nuclear extract was prepared from the cells and subjected to immunopurification using anti-FLAG antibody. Protein bands from SDS-PAGE analysis were analyzed by mass spectrometry and identified as indicated by arrows. The band at 52 kDa is a non-specific (nsp) product that was also observed in the control IP of a nuclear extract from non-transduced cells (not shown). Protein standards are shown on the left.

B. Reciprocal immunoprecipitations of the MLL-ENL/menin complex. (f)ME, or a mutant [(f)MEd] lacking the high affinity menin binding motif, were transiently expressed in 293T cells with (+) or without (-) HA-tagged menin. Nuclear extracts from the transfected cells were subjected to IP with anti-FLAG or anti-HA antibodies followed by immunoblotting with anti-FLAG, anti-HA, and anti-LEDGF (p75) antibodies.

C. MLL or MLL-p300 was transiently expressed with (+) or without (-) HA-tagged menin in 293T cells and subjected to IP with anti-HA antibody, followed by immunoblotting with anti-MLLN, anti-HA, and anti-LEDGF antibodies.

D. To detect endogenous MLL/menin/LEDGF association, nuclear extract of REH cells was subjected to IP with anti-menin antibody followed by immunoblotting with anti-MLLC, anti-menin, anti-LEDGF, and anti-ACTIN antibodies. Anti-Drosophila Myb (DmMyb) antibody was used as a negative control.

E. Schematic representations of the NUP98-LEDGF mutants are shown with a summary of their binding properties with MLL/menin on the right. The identified minimum MLL/menin binding domain (MmBD, residues 335-460) is indicated.

F. His-Express-Myc-tagged NUP98-LEDGF proteins were expressed in 293T cells and subjected to IP with anti-MYC antibody followed by immunoblotting with anti-Express epitope and anti-menin antibodies.