Fig 1: Expression of FMRP in Fmr1 I304N and WT littermates.(A) 50 ug of brain lysate from three Fmr1 I304N mice and three wild type littermates at P14, 2 mo, and 6 mo of age, were analyzed by Western blot for steady state FMRP expression using the anti-FMRP C-terminus antisera ab17722 (Abcam) and gamma-tubulin as a loading control. (B) Signal was quantified by chemiluminescence using Versadoc imaging and the percentage of I304N-FMRP signal compared to wild type FMRP is indicated. Error bars reflect the standard deviation of three mice. This experiment has been repeated several times and with different antibodies to FMRP (Chemicon ab2160 (1C3), 2F5 [84], and 7G1-1 [85] with consistent results. (C) 50 ug of brain lysate from P14 mice of the indicated genotype (littermates) was analyzed for expression of FMRP with ab17722, FXR1P, FXR2P, and gamma-tubulin. (D) 50ug of testes or spleen lysate from the same animals at P60 was analyzed in the same way with ab17722, and normalized to Hsp90 levels. (E) Brain lysates for polyribosome analysis were prepared from Fmr1 I304N mice and WT littermates, and fractionated over linear 20%–50% sucrose gradients. The levels of Fmr1 mRNA and Gapdh mRNA were quantified in each fraction by quantitative RT–PCR using the ΔΔCt method. An A254 absorption profile from one of the WT mice is shown in the upper panel and the 80S monosome and polyribosomes are indicated. Other gradients were indistinguishable within a WT and I304N littermate pair. Relative mRNA level in each fraction was plotted as a percentage of total mRNA to illustrate its distribution over the polyribosome gradient. Error bars reflect three technical replicates from a single littermate pair. The experiment has been repeated with additional littermate pairs, but cannot be plotted on one graph due to variable fraction collection between experiments. Representative graphs are shown.
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