Figure 3: GABABR1 and GABABR2 mRNAs are FMRP targets.
(a,b) RNA immunoprecipitation (RIP) for FMRP was performed using brains from wild type (WT) and Fmr1 KO male mice. (a) Gels showing RT–qPCR amplified product of input sample, FMRP RIP, and IgG control for GABABR1 and GABABR2. (b) Relative fold-enrichment as determined by real-time qPCR relative to input control (ΔΔCt=2−(Ct FMRP RIP−Ct IgG RIP)−(Ct FMRP input−Ct IgG input)). FMRP binds GABABR1, GABABR2, and the positive control CaMKIIα mRNA as detected in the RIP sample by real-time qPCR. Cacnα2δ2 served as a negative control and was not detected above background. WT: GABABR1=2.66±0.248, n=2; GABABR2=2.19±0.08, n=2; CaMKII=3.72±0.94, n=2; Cacnα2δ2=0.11±0.6, n=2. Fmr1 KO: GABABR1=0.01±0.0002, n=2; GABABR2=0.02±0.00006, n=2; CaMKII±0.04±0.01, n=2; Cacnα2δ2=0.012±0.005, n=2. (c–g) Western blot analysis of hippocampal synaptoneurosomes isolated from C57BL/6 WT and Fmr1 KO mice on a C57BL/6 background indicates the absence of (e) FMRP and increased protein expression of (f) GABABR1 and (g) GABABR2. Representative western blots are pseudocoloured to indicate intensity of bands, and the normalized optical density for each band is indicated below blot (Lookup table, below western blot). Western blots were normalized to the loading control, α-Tubulin. WT: FMRP=1.00±0.10; GABABR1=1.00±0.06; GABABR2=1.00±0.08. Fmr1 KO: FMRP=0.03±0.01; GABABR1=1.27±0.08; GABABR2=1.54±0.17. Experiment was repeated three times. Significance determined by Student’s t-test. Values represent mean±s.e.m. Uncropped versions of qPCR gel, with size markers, are available in Supplementary Fig. 7d. Uncropped version of western blots, with size markers are available in Supplementary Fig. 7b.
