Figure 7

PU.2 is a LTR-driven novel isoform of PU.1 that retains DNA-binding activity. Nuclear extracts were analyzed by EMSA using a radiolabelled probe containing the PU.1 DNA-binding consensus. (A) PU.1 and PU.2 expressed in COS cells co-migrate with bands observed in Klf3 ^−/− (KO) fetal liver nuclear extracts. PU.1 is supershifted by antibodies specific for the N-terminus and C-terminus, while PU.2 is only recognized by the C-terminal antibody. Nuclear extracts from COS cells transfected with empty pMT3 vector have been included as a control. (B, C) In Klf3 ^+/+ (WT) and Klf3 ^−/− (KO) fetal liver nuclear extracts, the band which co-migrates with PU.1 is recognized by both antibodies while the band that co-migrates with PU.2 is only supershifted by the C-terminal antibody, confirming the identities of the two bands. In (A-C), comparative quantification of nuclear extract preparations was achieved by western blotting for β-actin. (D) Nuclear extracts from untreated and tamoxifen-treated KLF1-ER inducible B1.6 cells. Nuclear extracts from COS cells transfected with PU.2 (and mock transfected) have been included as controls. The identity of the PU.2 is confirmed by addition of the C-terminal antibody (αPU.2). In (A-D), supershifts have been indicated by arrows, and additionally by an asterisk in (B).