VWRPY-containing AML proteins associate with Gro and TLE. (A) A scheme of 6× His AML proteins used in Far Western and pull-down analyses. Numbers on the 6× His derivatives indicate the regions that are included in the constructs. (B) In vitro association assay between AML and TLE proteins. Three micrograms of 6× His proteins: AML2, 33 kDa, lane 3; AML1b, 37 kDa, lane 4; AML1a, 39 kDa, lane 5; and control nonrelevant fused proteins, Bovine α1 and α2 PAF-AH (1b) subunits of 35 kDa, lanes 1 and 2, were analyzed by Western blotting and reacted with ³⁵S in vitro -translated TLE1. (C) Interaction in Jurkat cell extracts between immobilized AML and TLE. 6× His-AML1-b lane 5, AML2 lane 4 and control nonrelevant proteins: 6× His leptin lane 3 (18 kDa) and the α2 PAF-AH (1b) subunit lane 2 (35 kDa). Bound proteins were analyzed with pan-TLE monoclonal antibodies. Lane 1 AML1-b without Jurkat extract, served as control for nonspecific binding of anti-TLE antibodies to 6× His proteins. The size difference between in vitro-translated TLE (lane 6) and Jurkat extract TLE (lane 7) may result from expression of four different TLE genes in Jurkat cells. Ponceau staining are shown in the bottom panels of B and C.

TLE1 abrogates LEF- and β-catenin-dependent activity of TOPFLASH. 293 cells (0.5 × 10⁶) were transfected by CaPO₄ for 24 hr with 0.5 μg of reporter plasmids containing three LEF-1 binding sites (TOPFLASH) or three mutated LEF-1 binding sites (marked by × at the top panel, FOPFLASH), together with 2 μg of expression plasmid of LEF-1 and β-catenin, in the presence or absence of 2 μg of TLE1 expression vector.

Gro/TLE1 interacts specifically with AML1 and AML2 in the yeast interaction trap assay. Interactions between the AML coding regions fused to an activation domain (AD) and Gro/TLE1 proteins fused to LexA-DNA binding domain. LexA-β delineates LexA fused to Cbfβ. LexA-Dmcdc2 served as a negative control. The numbers indicate β-galactosidase activity resulting from reporter activation. + designates relative strength of blueness on X-gal indicator plates based on analyses of at least 8–10 colonies; ++ indicates >20 β-galactosidase units. − designates white colonies, indicative of absolute failure to activate the lacZ reporter gene. The assay configuration was dictated by the observation that LexA-AML constructs alone activated the reporter.

TLE1 repress TCRα and TCRβ activity in Jurkat cells. Jurkat cells (2.5 × 10⁶) were transfected by SuperFect for 7–8 hr with wild-type or mutated (marked by × at the top panel) TCRβ, 2 μg (A), or TCRα, 1 μg (B) reporters, with or without 1 μg of TLE1 expression vector. Data presented are the average of five (TCRβ) and six (TCRα) independent transfections carried out in duplicate. Control values of reporter lacking TCR enhancer (5–8%) were substracted. (C) A scheme summarizing the various interactions of AML/CBFα with the adjacently bound factors Ets, Myb, and C/EBP, as well as with the coactivators (arrows) p300/CBP and ALY, and with the negative regulators (bar) Ear 2 and TLE.

TLE1/Gro interactions with LEF-1/Pan. (A). TLE1 interacts with LEF-1 and not with β-catenin. In vitro association of ³⁵S-labeled TLE1 with LEF-1 or β-catenin that were immunoprecipitated (IP) from overexpressing 293 cells. Lanes 1 and 2: IP LEF-1 and β-catenin reacted with anti-HA antibody. Lanes 3 and 4: interaction of IP LEF-1 and β-catenin with ³⁵S-labeled TLE1. (B). Gro interacts with Pan. In vitro association between immobilized GST-Gro and in vitro-translated ³⁵S-labeled Pan or ΔPan lacking the first 50 aa. GST and GST-E(spl)m7 served as negative controls. Lanes 1 and 2: 10% of labeled Pan or ΔPan input, respectively. GST, GST-Gro and GST-E(spl)m7 incubated with labeled Pan were run in lanes 3,4, and 5, respectively; GST, GST-Gro, and GST-E(spl)m7 incubated with labeled ΔPan were run in lanes 6,7, and 8, respectively. (C) Pan (1–130) is adequate for interaction with Arm. In vitro association between immobilized GST-Pan (1–130) and ³⁵S-Arm. Lanes: 1, 10% of ³⁵S-Arm input; 2, GST; 3,GST-Pan (1–130); 4, GST-PanS25 (1–130). (D) Pan (1–130) is not adequate for the interaction with Gro. In vitro association between immobilized GST-Pan (1–130) and ³⁵S Gro. Lanes: 1, 10% of ³⁵S Gro input; 2, GST; 3, GST-Pan (1–130); 4, GST-PanS25 (1–130); 5, GST-E(spl)m7 as a positive control; 6, GST-E(spl)m7ΔWRPW-lacks the C-terminal tetrapeptide motif necessary for mediating m7-Gro interaction (21). Below B, C and D, are Coomassie blue-stained gels.