Figure 3.

Immunoprecipitation of phosphorylated MIXL1 by anti-phosphotyrosine antibody 4G10. Expression constructs pCMV2-flag-MIXL1 and pCMV2-flag-ΔCAD were transfected into HEK 293T cells. 500 μL of nuclear proteins (1μg/μL) from the transfected cells were precleared with 50 μL protein-A-agarose bead slurry (50%v/v) and incubated with 5 μg of the phosphotyrosine-specific antibody 4G10 or the antibody anti-V5 as an isotypic control overnight. The immune complexes were precipitated with 60 μL protein-A-agarose bead slurry (50%v/v). Washed pellets were dissociated and resolved in a NuPAGE gel for immunobloting with the antibody anti-MIXL1-N (1:500). The immunoprecipitation and immunobloting for full-length MIXL1 is shown in the left panel, while mutant ΔCAD in the right panel. A small fraction of both full-length MIXL1 and mutant ΔCAD were immunoprecipitated with the antibody 4G10 (Lane 2 or 6) but not with an isotypic control antibody anti-V5 (Lane 3, or 7). Supernatants from the immunoprecipitation reactions denoted by (-) show the integrity of proteins. A strong band below full-length MIXL1 appeared to be non-specific, as it was also present in the controls. The blot on the left (lanes 1, 2, 3) was exposed to a Kodak film for 30 seconds, while the blot on the right (lanes 4, 5, 6) was exposed for only 5 seconds.