To investigate whether Sp1, or any other member of this family, is expressed in HCECs, nuclear proteins were obtained from cells at P2 and incubated with a 5′-end–labeled oligonucleotide bearing the sequence of a high-affinity target site for Sp1. Formation of DNA-protein complexes was then monitored by EMSA. As shown on
Figure 1B , a number of DNA-protein complexes (designated
a–
d) could be observed when increasing amounts (5–20 μg) of HCEC crude nuclear proteins were added to the Sp1-labeled probe. Formation of both complexes
a and
d, and to some extent that of complex
b, was prevented by the addition of the unlabeled Sp1 competitor, but not by an oligonucleotide bearing the target sequence for the unrelated transcription factor NF1, providing evidence that formation of all three complexes (
a,
b, and
d) was specific
(Fig. 1C) . Further competitions using single-stranded oligonucleotides bearing the sequence from either the top (Sp1a) or the bottom (Sp1b) strand of the Sp1 target site revealed that formation of complex
d resulted from the recognition of the free-labeled Sp1b strand by a yet unknown nuclear protein. We next performed supershift experiments in EMSAs to define more precisely which of the Sp family members yield both complexes
a and
b. As revealed on
Figure 1D , formation of complex
a, but not that of any of the remaining complexes, was substantially decreased by the addition of a polyclonal antibody raised against Sp1, which also led to the formation of a new supershifted complex designated
a/Sp1Ab. The addition of an anti-Sp3 antibody resulted in only a moderate decrease in the formation of complex
a, but totally prevented formation of a weak complex designated
b*, often appearing on the gel just above complex
b when the concentration of the gel was lowered to 6%, leading to the formation of a new supershifted complex designated
a+b*/Sp3Ab
(Fig. 1D) . Altogether, these results suggest that complex
a is essentially made up of Sp1 with a small amount of Sp3 and that complex
b* results solely from the recognition of the Sp1-labeled probe by Sp3. Indeed, the addition of both the Sp1 and Sp3 antibodies to the reaction mix totally prevented formation of both complexes
a and
b*, as expected
(Fig. 1D) . That both Sp1 and Sp3 yield DNA-protein complexes which comigrated in EMSA is not unique and has been reported before.
4 44 45 The fast-migrating Sp3 complex yielding
b* apparently results from the recognition of the Sp1-labeled probe by an N-terminal truncated form of Sp3 that possess an intact DNA-binding domain but a partially deleted activation domain.
45 46 47 48 Formation of the
b complex, which in
Figures 1B and 1C could not be dissociated from complex
b*, appears to be nonspecific. No influence whatsoever has been observed on the formation of any of these complexes when the reaction was performed in the presence of the anti-Sp4 Ab, even when used at 4 μL (data not shown). In addition, the formation of neither complex
c or
d was altered by any of these antibodies.