Fig. 7. Methylation affects the ability of FMRP to bind specific mRNAs and regulate their translation. (A) Semi-quantitative RT-PCR of mRNAs associated with HeLa cell FMRP immunoprecipitates (IP) in the absence of AdOx, and following treatment of the cells with 20 µM AdOx for 24 hours prior to immunoprecipitation. Primer pairs for FMR1 mRNA, EF-1A mRNA, Tip60a mRNA, ßAPP mRNA and dynamin A1 were used to amplify each message from total HeLa cell RNA (T) and the FMRP IP (P) for each treatment. The five panels (left) show representative results for each message. The specific amplicons are labeled; non-specific amplicons and primer-dimers are marked by an asterisk. The graph (right) shows the percentage of the mRNA associated with the FMRP immunoprecipitate for AdOx-treated and non-treated cells. Values for three independent immunoprecipitations are shown. Asterisks indicate significant decreases in AdOx-treated cells (P<0.004 by ANOVA). (B) Comparison of the percent change in mRNAs associated with FMRP in AdOx-treated versus non-treated cells with the differences in in vitro binding of the mRNAs in the presence and absence of AdOx [results taken from Denman (Denman, 2002)]. (C) Western blot analysis of HeLa cell proteins from cells treated with 0 or 20 µM AdOx for 24 hours. Duplicate blots (left) were probed simultaneously with anti-FMRP mAb and anti-dynamin mAb, or anti-EF-1A mAb and anti-Hsp70cp mAb. The graph (right) shows the ratio of AdOx-treated to non-treated protein expression normalized to Hsp70cP. The number of determinations for each protein is shown in the bar. Double asterisk indicates that the AdOx-treated expression level is significantly different from the control (P<0.004, ANOVA); the single asterisk indicates that the AdOx-treated expression level is significantly different from the control (P<0.06, ANOVA).