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. 2013 Dec 11;42(5):3286–3297. doi: 10.1093/nar/gkt1272

Figure 5.

Figure 5.

Electrophoretic mobility shift assay with Mbb1-HA immunoprecipitates. (A) Binding of Mbb1-HA to the 5' UTR of psbB. A radiolabeled probe corresponding to the mature psbB 5' UTR was incubated with increasing amounts of Mbb1-HA immunoprecipitate (0.1, 1, 2.5 and 4.5 µg) and resolved by native polyacrylamide gel electrophoresis. Bound (B) and unbound (U) probes are marked. A similar experiment was performed with increasing amounts of Sbp-HA (sedoheptulose 1,7 bisphophatase) as a negative control. (B) Competition of RNA binding with WT or mutant psbB 5' UTR. One microgram of Mbb1-HA protein extract was incubated with a radiolabeled probe corresponding to the WT psbB 5' UTR and an excess (10-, 50- or 100-fold) of an unlabeled competitor corresponding either to the m26-31 psbB 5' UTR (left) or the WT psbB 5' UTR (right). (C) Binding of Mbb1-HA to the 5' UTR of psbH. A radiolabeled probe corresponding to the mature psbH 5' UTR was incubated with increasing amounts of Mbb1-HA immunoprecipitate as in (A). (D) Competition of RNA binding with WT or mutant psbH 5' UTR. Competition was performed as in (B) with WT or mutant (ApaS) psbH 5' UTR unlabeled RNA.