p14 NF-E4 binds CP2 but not p22 NF-E4 in vivo. (A) Coimmunoprecipitation studies of CP2 and the NF-E4 isoforms. 293T cells were transduced with the MSCV p14 NF-E4-FLAG retrovirus alone (lane 1) or in combination with a pCAGGS vector containing the p22 NF-E4 cDNA tagged at the 5′ end with an HA epitope (HA-p22 NF-E4) (lane 2) or the CP2 cDNA tagged at the 3′ end with an HA epitope (CP2-HA) (lane 3). Cell extract from the 3 lines was immunoprecipitated with anti-FLAG antisera, and the precipitates were fractionated by SDS-PAGE and blotted with antisera to HA or FLAG as indicated (top panels). The input extracts for these experiments were also immunoblotted with antisera to HA and FLAG as loading controls (bottom panels). The migration of CP2-HA, HA-p22 NF-E4, and p14 NF-E4-FLAG are indicated. (B) Coimmunoprecipitation of endogenous CP2 and p14 NF-E4 from K562 cells. Nuclear extract from K562 cells was immunoprecipitated with polyclonal antiserum to CP2 (lane 3) or preimmune serum (lane 2) and the precipitates fractionated by SDS-PAGE prior to blotting with antiserum to NF-E4 (top panel) or CP2 (bottom panel). The first lane shows the levels of p14 and CP2 in the input extract.