Fig. 5. Kaiso interacts directly with Importin-2. (A) Equivalent amounts of immobilized GST, GST/Importin- and GST/Importin- + 6x-His/Importin-ß were incubated with radioactively labeled, in vitro transcribed and translated (IVTT) wild-type Kaiso and NLS-mutated Kaiso, as indicated. Wild-type Kaiso but not NLS-mutated Kaiso was able to bind GST/Importin- in both the presence and absence of 6x-His/Importin-ß. The autoradiograph of samples after 7% SDS-PAGE is shown. SDS-PAGE of 2.0 µl IVTT proteins is shown as representing 20% total input. (B) To determine whether Kaiso and Importin-2 form a complex in vivo, we performed coimmunoprecipitation experiments from HCT116 cell lysates with Kaiso- and Importin-2-specific monoclonal antibodies. Importin-2 co-precipitated weakly with endogenous Kaiso (lane 1) but not with p120^ctn (lane 2) or the irrelevant negative control anti-HA antibody 12CA5 (lane 4).