N-terminal MLL interacts with C-terminal MLL.

Copurification of MLL^C with MLL^N. (A) BOSC23 cells were transfected with pLNCX vectors encoding MLL or the DDAA mutant tagged with FLAG at their N-termini. Proteins were immunoprecipitated with anti-FLAG antibody, eluted by incubation with FLAG peptide, separated in a denaturing gel, and stained with Coomassie brilliant blue (CBB). The purified fragments are indicated by asterisks (MLL^N and MLL^C in lane 1, unprocessed MLL in lane 2). Molecular weight markers are on the left. (B) BOSC23 cells were transfected with pLNCX vector alone or pLNCX encoding MLL protein tagged with His and FLAG at its N-terminus and HA at its C-terminus [denoted (Hi-FL)MLL(HA)]. Cell lysates were immunoprecipitated with anti-FLAG antibody or anti-HA antibody. The whole cell lysates (WCE) and the immunoprecipitates (denoted as IP:anti-HA and IP:anti-FL) were analyzed by immunoblot with anti-His antibody (left panel) or anti-HA antibody (right panel). The purified fragments are indicated by asterisks (MLL^N in left panel, MLL^C in right panel). (C) Interaction of endogenous MLL^N and MLL^C. Cell lysates were immunoprecipitated with nonspecific mouse IgG, mouse anti-MLL N-terminal (mmN4), or anti-MLL C-terminal (mmC2) antibodies. The precipitates were analyzed by immunoblot with a mouse anti-MLL C-terminal (mmC2) antibody. MLL^C is indicated by an asterisk.