Binding of Sp1 to the –82/+41
region. EMSA were carried out using nuclear extracts from Hep3B
or A6 cells that had been transfected with the pCS2+ control
vector (con) or the pCS2+xα expression
plasmid (α-tran) and either –82/+41
promoter fragment (A and B) or
an oligonucleotide containing the putative Sp1 recognition sequence
(C). Competition experiments used a 500-fold excess
of oligonucleotides for: a high affinity C/EBP binding
site (+D-C/EBP), consensus Sp1 recognition sequence
(+SP1), consensus USF binding site (+USF), sequences
corresponding to the –53 to –81 region of the
xC/EBPα promoter (+NS),
the Sp1 site from the xC/EBPα promoter
(+wt-SP1) or a larger sequence containing deletion of this
site (+del-SP1). In (C), + indicates
the profile obtained with extracts from pCS2+xα-transfected
cells in the presence of antiserum against Sp1. Vertical lines labelled
B, ss and F show the position of the DNA–protein complex,
the antibody–DNA–protein-supershifted complex
and the free probe, respectively.