Electrophoretic mobility shift assays (EMSAs) of fragments within the first 89 bps of the LF promoter.

(A) An EMSA was performed by using double-stranded phosphorus 32–labeled LF −89 bp to −66 bp (SP1A) as a probe with nuclear extracts from U937 cells. Binding of Sp1 (lower arrow) was subjected to competition with a 100-fold molar excess of self cold competitor (CC) (lane 3), Sp1 consensus probe (lane 4), and a CD18 Sp1 cold competitor (lane 5) but not a 100-fold molar excess of a nonspecific (n.s.) probe (lane 6). Preincubation of Sp1A probe and U937 nuclear extracts with antiserum to Sp1 resulted in a band shift (lane 7, upper arrow [supershift]). Purified Sp1 protein also bound to the Sp1A probe (lane 8). (B) Results of EMSA analysis using nuclear extracts from uninduced (U937/U) and zinc-induced (U937/I) U937-C/EBPα cells and LF −53 bp to −35 bp (SP1B) as a probe. In both uninduced and induced extracts, Sp1 was eliminated competitively by the addition of a 100-fold molar excess of Sp1B probe (self CC, lanes 3 and 7) and an Sp1 consensus probe (lanes 4 and 8) but not by a nonspecific probe (n.s., lanes 5 and 9, lower arrow). Preincubation of protein-DNA complexes with anti-Sp1 antiserum resulted in a supershifted band (upper arrow) in both uninduced (lane 9) and induced (lane 10) nuclear extracts. (C) EMSA supershift analysis was performed on the radiolabeled LF-C/EBP binding site (LF −74 bp to −51 bp) by using nuclear extracts from uninduced U937-C/EBPα cells and from U937-C/EBPα cells induced with zinc. C/EBPα bound to the LF-C/EBP site from both uninduced (lane 2) and induced (lane 5) U937-C/EBPα extracts (lower arrow). The protein-DNA complex was supershifted (upper arrow) by anti-C/EBPα antiserum (lanes 3 and 6) but not by anti-C/EBPβ antiserum (lanes 4 and 7).