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. Author manuscript; available in PMC: 2013 Jun 17.
Published in final edited form as: Biochem J. 2010 Jul 1;429(1):215–224. doi: 10.1042/BJ20100286

Figure 2. Efficient transcription in the Cat-1 gene promoter is mediated via a Sp1-binding site.

Figure 2

(A) Diagramatic representation of constructs 13 and 13m as used in the present study. (B) C6 cells were transfected with 100 ng of construct 13 or 13m without (CONTROL) or with 100 ng of Sp1 expression plasmids in cells plated in six-well plates. The total amount of transfected DNA was kept constant with the addition of non-specific DNA (pcDNA 3.1). LUC assays were performed 48 h post-transfection and values were normalized to protein content. Results are means ± S.E.M. expressed as the ratio of 13/13m. *P < 0.05 between Sp1 and CONTROL samples. (C) Cat-1 mRNA levels in Sp1−/− and Sp1+/+ cells. Results from qRT-PCR analysis of total RNA using gene-specific primers were normalized to the GAPDH mRNA signal. (D and E) EMSAs were performed by incubating a 32P-labelled double-stranded oligonucleotide containing the Sp1-binding site of the Cat-1 gene with nuclear extracts from (D) C6 cells or (E) cells expressing recombinant Sp1. Competition assays were performed with unlabelled Cat-1 (WT) or Cat-1 mutant (Sp1 MUT) oligonucleotides as indicated. (F) ChIP was performed using C6 cells with antibodies against Pol II and Sp1. Samples without antibody (-Ab) or with normal rabbit IgG were used as negative controls. PCR was performed with primer sets specific for the regions of interest (Cat-1 promoter or exon 13). (G) Cat-1 mRNA levels in Sp1−/− and Sp1+/+ either untreated amino-acid-fed (CON), amino-acid-starved (Starved) or thapsigargin (Tg)-treated for the indicated time. The CON condition involved serum-containing medium that was used to grow the Sp1 WT and Sp1−/− cells (see the Experimental section). Thapsigargin treatment was performed in the same medium, as described in the Experimental section. The amino-acid-starved condition was employed by incubating the cells in KRB supplemented with 10 % (v/v) dialyzed FBS. (B, C and G) Results are means ± S.E.M. for three independent experiments; (D–F) a representative gel from three independent experiments is shown.