Figure 4

Gag-binding activity of artificial ankyrins. (A), Competition ELISA. Samples of biotinylated Gag-binders Ank^GAG1B8, Ank^GAG1D4 and Ank^GAG6B4, and of control biotinylated αRep-A3-binder Ank^A32D3 were mixed with their corresponding non-biotinylated form (black bars), or mixed with irrelevant soluble target (grey bars), or mixed with buffer containing no inhibitor (white bars). Mixtures were added to H₆MA-CA- or αRep-A3-coated wells, as indicated at the bottom of the panel. Bound-ankyrins were detected by addition of HRP-conjugated extravidin, followed by the TMB substrate. (B), Far Western blotting. Lysates of BV-H₆MA-CA-infected Sf9 cells were electrophoresed in SDS-gel, proteins transferred to a PVDF membrane, and membrane cut into strips. Gag-binding activity was determined by incubation of the strips with the different biotinylated ankyrins Ank^GAG1B8, Ank^GAG1D4, Ank^GAG6B4, and Ank^A32D3, as indicated on top of the strips. On the rightmost strip, the respective positions of the Gag proteins H₆MA-CA and H₆MA were determined using anti-histidine tag antibody (arrowheads). (C), Indirect ELISA. H₆CA was captured on nickel-coated plate, and used as substrate for binding assay of biotinylated ankyrins Ank^GAG1B8, Ank^GAG1D4, Ank^GAG6B4, and Ank^A32D3. Bound-ankyrins were quantitated as in (A). BG, background signal.