Western blot analyses of HRX proteins using various antibodies. (A) Western blot analysis of flag-tagged HRX-ENL expression. Lanes 1 through 4 contain a nuclear extract of Bosc 293 cells transfected with either the N-terminal flag-tagged HRX-ENL (H) or vector alone (V) (2.5 μg protein per lane). Lanes 1 and 2 were probed with MoAb HRX 107 whereas lanes 3 and 4 were probed with the antiflag antibody. Arrow indicates the protein detected by both MoAb HRX 107 and the antiflag antibody. For lanes 5 and 6, extracts of Bosc 293 cells (75 μg) that had been transfected with N-terminal flag-tagged HRX-ENL (H) or vector alone (V) were immunoprecipitated with MoAb HRX 107, with subsequent Western blotting with the antiflag antibody. (B) Comparison of MoAb HRX 107 with polyclonal anti-HRX and anti-ENL antibodies. Each lane contains extracts of Bosc 293 cells transfected with an HRX-ENL expression construct (H) or vector alone (V) as indicated above the gel lanes. Protein amounts applied to the gel were as follows: 50 μg in lanes 1, 2, and 6; 10 μg in lane 5; 20 μg in lanes 3, 4, 7, and 8. Note that the negative control lane 6 in the Western blot probed with MoAb HRX 107 contained five times as much protein as lane 5. Arrow indicates the protein detected by MoAb HRX 107 and the polyclonal antibodies αHRX1, αHRX2, and αENL. Arrowhead indicates the smaller isoforms of HRX-ENL detected by the polyclonal antibodies αHRX1 and αENL. (C) Western blot analysis of whole-cell lysates from Molt-4 (T-cell acute lymphoblastic leukemia), and two cell lines (HB 11; 19 and RS 4; 11) expressing chimeric HRX fusion proteins using HRX 107 on a 5% SDS-PAGE gel.