Supershift EMSA in the presence of a c-Myb-specific antibody. (A). Nuclear extracts (5 μg) from K-562 cells were incubated with the radiolabeled GS945 probe (2.4 ng) representing the 21-bp triplication from the FeLV-945 LTR. Shown are probe only (lane 1), complex formation in the presence of nuclear extract (lane 2), and complex formation in the presence of 200-fold molar excess of non-specific (lane 3) or specific competitor (lane 4). Reaction performed in the presence of monoclonal antibody to c-Myb (4 μg) resulted in supershift of the specific complex (lane 5) which was not observed in the presence of 200-fold molar excess of specific competitor (lane 6). (B). Lanes 1, 2 and 3 represent repetitions of lanes 1, 2 and 5 of (A). Reaction with a isotype control antibody (lane 4) did not result in supershift. Indicated are the specific complex (solid arrow), non-specific complexes (open arrows), and the supershifted complex (asterisk). (C). EMSA performed using the radiolabeled GS61E probe, which contains only a single copy of the 21-bp element. Shown are probe only (lane 1), reaction performed in the presence of K-562 nuclear extract (5 μg; lane 2), and reaction performed in the presence of 100-fold molar excess of unlabeled GS945 (lane 3), GS61E (lane 4) or non-specific competitor (lane 5). The absence of complex formation using the GS61E probe demonstrates the requirement for the 21-bp triplication.