MudPIT analysis of the proteins interacting with truncations of the mixed lineage leukemia (MLL/KMT2A)

ABSTRACT: Plasmids encoding Halo-tagged mixed lineage leukemia (MLL)/ histone-lysine N-methyltransferase (KMT2A) proteins were transiently transfected into HEK293 cells. Four truncations of MLL were also expressed into HEK293 cells: MLL-CT, which only contains the C-terminus of MLL (2760-3972aa); MLL-Inter, which is lost in MLL chimeras due to chromosome translocation (T1: 1163-2158aa); a truncation of the N-terminus to the second PHD finger (T2: 1478-2158aa); and another truncation to the third PHD domain (T3: 1545-2158aa). Two days later, HEK293 cells were harvested and lysed with mammalian lysis buffer (Promega). Halo-tagged proteins were purified with HaloTag resin in the presence of Benzonase (Sigma) and eluted with TEV protease. The eluates were precipitated with TCA. After washing with acetone, the protein mixtures were digested with endoproteinase Lys-C and trypsin (Roche) and analyzed by MudPIT. Vector only (Halo Vector) transfected cells were used as negative controls. The Halo-tagged MLL internal region (MLL-Inter) was also purified from HEK293 cells treated with DMSO or interleukin 1 receptor associated kinase (IRAK1) inhibitor for 24 h. Two biological replicate analyses were performed.

INSTRUMENT(S): LTQ

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Laurence Florens 

PROVIDER: MSV000080298 | MassIVE | Thu Oct 27 08:07:00 BST 2016

SECONDARY ACCESSION(S): PXD005233

REPOSITORIES: MassIVE