Primer extension analyses of directed hydroxyl radical probing experiments to test for disruption of the interaction of mutated RBDs with the EMCV and PV IRESs. Assays of the designated PTB′ mutants with the EMCV IRES are shown in panels A–H, with panels A,C,E,G examining interactions with Domains D–F, and panels B,D,F,H, the interactions with Domains G–K. Assays with the PV-1 IRES are in panels I–L. The cleavage products arising from Fe(II) conjugated to the relevant non-mutated RBD are indicated by vertical lines to the left of the reference standard lanes. The inactivating mutations analyzed in each subpanel are as follows: A,B,I—RBD1 mutations; C,D,J—RBD2 mutations; E,F,K—RBD3 mutations; and G,H,L—RBD4 mutations. Lanes A,C,G are from sequencing ladders with the same primers as the primer extension lanes. The arrowheads to the left of each panel indicate the position of the reference standard cleavage site in the IRES.
