Structural and biochemical properties of wild-type and mutant PKNOX1 proteins. (A) Schematic representation of the mutants used in this study. Numbers above the constructs indicate amino acid positions. HM^A/HM^B indicates Homothorax-Meis domain A/B; M-CTD, MEIS1 CTD; and P-CTD, PKNOX1 CTD. (B) Western blot analysis of FLAG-tagged wild-type and mutant PKNOX1 expression in cytoplasmic (top) and nuclear (middle) lysates of GP-E + 86 producer cells infected with retroviruses expressing the indicated constructs. Tubulin-α levels are shown as a loading control (bottom panel). (C) Immunoprecipitation of lysates from NIH-3T3 cells transfected with the constructs indicated above the horizontal line was carried using anti-HA or an isotypic antibody, as shown above the top panel. The amount of interacting FLAG-tagged mutants was determined by Western blot analysis using anti-FLAG antibody (top panel), whereas the amount of precipitated HA-tagged PBX1A was determined by anti-HA antibody (bottom panel). (D) Immunoprecipitation of lysates from NIH-3T3 cells transfected with the constructs indicated above the horizontal line was performed using anti-FLAG or isotypic antibody, as shown. The amount of interacting HA-tagged PBX1A was determined using anti-HA antibody. Input level is shown in lane 1 and corresponds to 30 μg of protein.