Fig. 3.

Shift assays of eB mutants (Mut) with conserved secondary structure and competition of eB-mediated shifts with each mutant. A: shift assays of eB mutants with conserved secondary structure. Lane 1, eB probe (negative control); lanes 2 and 18, eB riboprobe incubated with cytoplasmic NCI-H441 extract (positive controls); lane 3, competition of eB-mediated shifts with 200-fold molar excess of eB unlabeled RNA; lanes 4–17, mutant riboprobes incubated with extracts in the presence (+) or absence (−) of unlabeled WT eB 200-fold molar excess. Mut 1 and 2 formed shifts similar to the WT eB that were competed with excess WT eB unlabeled probe. Mut 1 also formed nonspecific shifts that were not competed with WT eB (nonspecific interactions). Mut 3 and 4 failed to form shifts. Mut 5 gave rise to the upper shift band (*) of the WT eB, whereas Mut 6 formed the lower shift band (circled) of the WT eB. B: competition assays of eB-mediated shifts, with 200-fold molar excess of mutant probes with conserved secondary structure, and excess AD and ABD unlabeled RNAs. Lane 1, eB probe (negative control); lanes 2 and 13, eB riboprobe incubated with cytoplasmic NCI-H441 extract (positive controls); lane 3, competition of eB-mediated shifts with unlabeled eB RNA; lane 4, competition of eB-mediated shifts with unlabeled random (R) RNA; lanes 5–10, competition of eB-mediated shifts with mutant unlabeled RNAs; lane 11, competition with unlabeled AD RNA; lane 12, competition with unlabeled ABD RNA; lane 14, R probe; lane 15, R biotinylated probe incubated with cytoplasmic NCI-H441 extract.