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Figure 4 | BMC Molecular Biology

Figure 4

From: A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter

Figure 4

Evaluations for DNA-binding capacities of various HPV-2 E2 proteins with oligo HPV-E2BS by EMSA. A. Specificity assays of the molecular binding of oligo HPV-E2BS with recombinant HPV-2 E2. Left panel: Competition assays. 250 fM biotin-labeled oligonucleotide probe HPV-E2BS were mixed with 1 μg recombinant protein HPV-2 E2-FL, competed with 50-, 100-, and 500-fold excesses of homologous oligo (HPV-E2BS) and 500-fold excesses of heterologous oligo (T7). Oligonucleotide T7 represents E. coli T7 promoter-specific double-stranded sequences (5'-TCGATAATACGACTCACTATAGGGAGAAGATC-3'). Right panel: Supershift assay with mAb anti-HPV-2 E2. 7 μg recombinant HPV-2 E2-FL was incubated with 1 μl mAb against HPV-E2 at RT, prior to mixing with 250 fM biotin-labeled probe HPV-E2BS. The E2-DNA complexes and the supershifts are indicated by arrows. F: free probe. B. EMSA of various constructs of E2-C. C. EMSA of various constructs of E2-HC. D. EMSA of various constructs of E2-FL. 250 fM biotin-labeled probe HPV-E2BS were mixed with different amounts of various E2 proteins. The concentrations of various E2 protein were indicated in the bottom of the graphs. The protein-DNA complexes were separated in 6.5% PAGE gels (Left) indicated by arrows marked E2-FL, E2-HC and E2-C, respectively. The competition assays were performed in the presences of 500-fold excess of unlabeled probe HPV-E2BS prototype (Middle). F: free probe. The binding capacities of various E2 proteins (Figure 4B, C and D) were evaluated by densitometric quantification of the signal of each complex with computer-assisted software Image TotalTech. The average values are calculated from three independent tests and presented with as mean ± SD (Right)

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