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. 2012 Jun 7;40(16):7844–7857. doi: 10.1093/nar/gks544

Figure 2.

Figure 2.

DNA-binding activity of endogenous Sp1, Sp2 and Sp3 in nuclear extracts. (A) Expression of Sp1, Sp2 and Sp3 in MEFs. Nuclear extracts of wt MEFs (wt), Sp3-deficient MEFs (Sp3ko), MEFs with floxed Sp2 alleles (Sp2cko) and Sp2-deficient MEFs (Sp2ko) were resolved by SDS–PAGE and successively subjected to western blot analysis for Sp1, Sp2 and Sp3 (Sp3li, long Sp3 isoforms; Sp3si, short Sp3 isoforms). Mi2 was used as a loading control. (B) EMSA with nuclear extracts of wt MEFs. Extracts were incubated with 0.2 ng of 32P-labelled GC oligonucleotide. Antibodies against Sp1, Sp2 and Sp3 were included in the binding reactions as indicated. Sp1, Sp3li and Sp3si containing DNA protein complexes are indicated on the right side. (C) Nuclear extracts of MEFs with the indicated genotype, and of HEK293 and HeLa cells were analysed by DAPA using a biotinylated GC box oligonucleotide (GC) and an oligonucleotide with a mutation in the Sp recognition sequence (GCm). Bound proteins were successively analysed for the presence of Sp2, Sp1 and Sp3 proteins by western blotting. IN, input 15%; B, bound protein 80%. The asterisk marked usSp1 next to the Sp3 blot of HEK293 and HeLa cells denotes unstripped Sp1 signals that were removed incompletely prior to the re-probing for Sp3.