FIGURE 3.

C/EBPβ and Hipk2 interact in untransfected cells and in the absence of other eukaryotic proteins. A, shown is an in vitro pulldown experiment. Similar amounts of bacterially expressed GST and GST-Hipk2-(70–220) fusion protein were bound to glutathione-Sepharose, incubated with bacterially expressed C/EBPβ, and subjected to a GST pulldown assay. Input (12% of the total amount, lane 1) and bound proteins (lanes 2 and 3) were analyzed by SDS-PAGE and Western blotting using C/EBPβ-specific antibodies. The bottom panel shows a Coomassie Blue-stained gel of the bacterial proteins to confirm equal loading of the Sepharose beads. B, total cell extract of untransfected PC3 cells was immunoprecipitated with a monoclonal antibody against human C/EBPβ (lane 3) or with the monoclonal anti-HA-antibody (lane 1). Lane 2 shows an aliquot (5%) of the total cell extract (TCE). Lane 4 shows a shorter exposure of lane 2. All samples were analyzed by Western blotting with polyclonal Hipk2-specific antiserum.