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Fig. 2. Methylation affects the protein-protein interactions of FMRP. (A) Methylated FXR1P but not non-methylated FXR1P heterodimerizes with FMRP in vitro. Methylated and non-methylated FXR1P was bound to equal amounts of biotinylated-FMRP. Heterodimers were isolated by affinity capture on SoftLink resin. The unbound (U) and bound (B) fraction for each reaction was resolved by SDS-PAGE, blotted and probed with anti-FXR1P pAb. As a control, FXR1P binding to SoftLink resin in the presence of a mock RRL reaction without FMRP plasmid (Mock) is shown. The far right-hand panel shows that the anti-FXR1P pAb does not detect biotinylated-FMRP under these conditions. (B) The percent binding of ³⁵S-FXR1P to biotinylated-FMRP produced in the presence of various concentrations of AdOx is plotted. Binding to FMRP produced in the presence of 3 µM AdOx was significantly less than in its absence (P<0.01, ANOVA). Binding to FMRP produced in the presence of 6 µM AdOx was also significantly less than in its absence (P<0.006, ANOVA). The number of determinations for each concentration is shown in the bar. (C) Methylated FMRP but not non-methylated FMRP heterodimerizes with FXR1P in vitro. Biotinylated-methylated and biotinylated-non-methylated FMRP produced in RRL was bound to equal amounts of methylated-FXR1P. Heterodimers were isolated and detected as described in A. For controls, FXR1P binding to SoftLink resin in the presence of a mock RRL reactions without FMRP plasmid and with or without AdOx (Mock and Mock+AdOx) is shown. (D) The percent binding of ³⁵S-FMRP to biotinylated-FMRP produced in the absence or presence 6 µM AdOx is plotted. The number of determinations for each concentration is shown in the bar.

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