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Fig. 3. In vitro DNA-binding assay of maize and Arabidopsis B-class proteins. (A) Autoradiogram of in vitro transcribed and translated SI1 and ZMM16 proteins incubated with labeled AGL5 CArG-box probe. Neither SI1 nor ZMM16 alone are capable of binding the AGL5 CArG-box, but together (SI1+ZMM16 lane) they can, as indicated by the mobility shift of labeled AGL5 CArG probe. Control lane consists of TNT lysate (without added plasmid DNA) incubated with probe. FP designates the lane loaded only with free probe. Arrows indicate background bands in the negative control caused by nonspecific binding of lysate proteins to the probe. (B) As in A, but the probe contains mutations in the AGL5 CArG-box that abolishes SI1-ZMM16 heterodimer binding (see Materials and methods for details). (C) As in A, but includes in vitro transcribed and translated AP3 and PI proteins. Weak binding to the probe in lanes containing PI (AP3+PI and PI+SI1) is due to poor in vitro expression of the PI template, as demonstrated by ³⁵S-labelled TNT control reactions (data not shown).

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