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. 2003 Apr;23(8):2720–2732. doi: 10.1128/MCB.23.8.2720-2732.2003

FIG. 7.

FIG. 7.

Functional significance of HMGI-Y, Sp1, and C/EBPβ for IR gene transcription in MCF-7 cells and in HeLa nuclear extract (NE) immunodepleted of HMGI-Y and endogenous Sp1 gene activators. (A) MCF-7 cells were transiently transfected with the pCAT-C2 reporter vector (5 μg) plus increasing amounts (1, 5, or 10 μg) of HMGI-Y and Sp1 expression plasmids, either alone or in combination. (B) CAT activity in MCF-7 cells is reduced by decoy oligonucleotides with the C/EBPβ binding sequence and is eliminated by transfection of a pCAT-C2 reporter construct carrying mutated binding elements for HMGI-Y and/or Sp1. In either case, data represent the means ± standard errors for three separate experiments; values are expressed as factors by which induced activity increased above the level of CAT activity obtained in transfections with pCAT-C2 control vector alone, which is assigned an arbitrary value of 1. Immunoblots of HMGI-Y and Sp1 in each condition are shown in the autoradiograms. (C) Immunoblot analyses of HeLa NE with polyclonal antibodies against HMGI-Y and Sp1. Extracts were not treated (NT) or subjected to one, two, or three rounds of depletion with immobilized anti-HMGI-Y and anti-Sp1 antibodies or a control (unrelated rabbit serum immunoglobulin G [IgG]) antibody. (D) Transcription of the IR gene in immunodepleted extracts during 1 h of incubation at 30°C with the IR-CAT DNA as the template DNA in the absence or presence of the indicated proteins (100 ng each). Transcripts were identified by primer extension with a CAT primer and resolved on a 8% denaturing polyacrylamide gel. An activation value of 1 denotes the basal level of transcription in the absence of activators. A representative of three separate assays is shown. (E) Transcription of the IR gene was performed as in panel D, except that increasing amounts (0, 50, and 100 ng) of either Sp1 or HMGI-Y were used in the presence of a fixed amount (100 ng) of HMGI-Y or Sp1, respectively. (F) IR gene transcription was measured in untreated HeLa NE, in the absence or presence of oligonucleotides (10 ng each) bearing either a wild-type (wt) or mutant (m) binding sequence for HMGI-Y, Sp1, or C/EBPβ.