Figure 5
Figure 5. The interaction between MLL and AML1 is impaired by some mutant AML1 proteins found in leukemia patients. (A) Diagram of a series of the N-terminal frame shift and misssense mutations. The black bar indicates the MID. The black area indicates the Runt-domain. The black x's indicate missense mutation of AML1 found in MID (details in Figure 5C). (B) The AML1 frame shift mutants interact with MLL and block the interaction between wild-type AML1 and MLL. Lane 1, transfection control; lane 2, AML1 with vector control; lane3, AML1 with AML1 (1-57); lane 4, AML1 with AML1 (1-72); lane 5, AML1 with AML 1(1-91); and lane 6, AML1 with AML1 (1-105). Western blots were done using indicated antibodies (row 1, anti-AML1; row 2, IP with anti-Flag and IB with anti-Flag; row 3, IP with anti-Flag, running with 10% SDS-PAGE gel and IB with anti-AML1; and row 4, IP with anti-Flag, running with 20% SDS-PAGE gel and IB with anti–AML1-N). (C) The N-terminal missense mutations of AML1 impair MLL interaction. Lane 1, transfection with pCS2 vector control and AML1; lane 2, MLL with AML1; lane 3, MLL with AML1 (L29S); lane 4, MLL with AML1 (A33V); lane 5, MLL with AML1 (G42R); lane 6, MLL with AML1 (R49H); lane 7, MLL with AML1 (R49S); lane 8, MLL with AML1 (H58N); lane 9, MLL with AML1 (V63A); lane 10, MLL with AML1 (S67I); and lane 11, MLL with AML1 (W79C). Western blots were done using indicated antibodies (row 1, anti-AML1; row 2, IP with anti-Flag and IB with anti-Flag; and row 3, IP with anti-Flag, IB with anti-AML1). (D) ChIP assay with anti-MLL antibodies and with primer set at 3′URE region of PU.1 gene (Figure 2A primer set 2) with 416B stable cell lines: 1, pBEX; 2, pBEX-AML1; 3, pBEX-AML1 (1-91); 4, pBEX-AML1 (1-105); 5, pBEX-AML1 (L29S); 6, pBEX-AML1 (H58N); 7, pBEX-AML1 (R139Q); and 8, pBEX-AML1 (R177Q). (E) ChIP assay with anti-H3K4me3 antibodies and with primer set at 3′URE region of PU.1 gene (Figure 2A primer set 2) in 416B stable cell lines in experiments 5D. (F) Real-time PCR assay for PU.1 expression in 416B stable cell lines in experiments in panels D and E.

The interaction between MLL and AML1 is impaired by some mutant AML1 proteins found in leukemia patients. (A) Diagram of a series of the N-terminal frame shift and misssense mutations. The black bar indicates the MID. The black area indicates the Runt-domain. The black x's indicate missense mutation of AML1 found in MID (details in Figure 5C). (B) The AML1 frame shift mutants interact with MLL and block the interaction between wild-type AML1 and MLL. Lane 1, transfection control; lane 2, AML1 with vector control; lane3, AML1 with AML1 (1-57); lane 4, AML1 with AML1 (1-72); lane 5, AML1 with AML 1(1-91); and lane 6, AML1 with AML1 (1-105). Western blots were done using indicated antibodies (row 1, anti-AML1; row 2, IP with anti-Flag and IB with anti-Flag; row 3, IP with anti-Flag, running with 10% SDS-PAGE gel and IB with anti-AML1; and row 4, IP with anti-Flag, running with 20% SDS-PAGE gel and IB with anti–AML1-N). (C) The N-terminal missense mutations of AML1 impair MLL interaction. Lane 1, transfection with pCS2 vector control and AML1; lane 2, MLL with AML1; lane 3, MLL with AML1 (L29S); lane 4, MLL with AML1 (A33V); lane 5, MLL with AML1 (G42R); lane 6, MLL with AML1 (R49H); lane 7, MLL with AML1 (R49S); lane 8, MLL with AML1 (H58N); lane 9, MLL with AML1 (V63A); lane 10, MLL with AML1 (S67I); and lane 11, MLL with AML1 (W79C). Western blots were done using indicated antibodies (row 1, anti-AML1; row 2, IP with anti-Flag and IB with anti-Flag; and row 3, IP with anti-Flag, IB with anti-AML1). (D) ChIP assay with anti-MLL antibodies and with primer set at 3′URE region of PU.1 gene (Figure 2A primer set 2) with 416B stable cell lines: 1, pBEX; 2, pBEX-AML1; 3, pBEX-AML1 (1-91); 4, pBEX-AML1 (1-105); 5, pBEX-AML1 (L29S); 6, pBEX-AML1 (H58N); 7, pBEX-AML1 (R139Q); and 8, pBEX-AML1 (R177Q). (E) ChIP assay with anti-H3K4me3 antibodies and with primer set at 3′URE region of PU.1 gene (Figure 2A primer set 2) in 416B stable cell lines in experiments 5D. (F) Real-time PCR assay for PU.1 expression in 416B stable cell lines in experiments in panels D and E.

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