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. Author manuscript; available in PMC: 2019 Sep 16.
Published in final edited form as: Cell Rep. 2019 Sep 3;28(10):2581–2593.e5. doi: 10.1016/j.celrep.2019.08.001

Figure 2. dFMRP Is Required to Limit Wnd Levels in the Nervous System and Binds Wnd mRNA, Suggesting Translational Repression.

Figure 2.

(A) Representative western blots of Wnd, dFMRP, and Tubulin from larval VNCs taken from wild type, two different dFmr1-transheterozygous mutants, and a dFmr1 transheterozygous mutant with a wild-type dFmr1 genomic rescue construct inserted on a different chromosome.

(B) Quantification of mean (±SEM) Wnd levels in each genotype. Wnd levels were increased in both dFmr1 mutants, and the genomic rescue of dFmr1 abolished the increase.

(C) qPCR quantification of wnd mRNA levels in wild-type versus dFmr1 mutants, presented as fold change relative to wild type. The wnd mRNA levels did not differ between wild-type and dFmr1 mutants.

(D) Representative western blots of wild-type and dFmr1 mutant larval VNCs after ex vivo incubation in cycloheximide (CHX) for the indicated times. Wnd levels are higher at baseline in dFmr1 mutants, but protein degradation is not inhibited.

(E) Quantification of mean (±SEM) Wnd levels in each genotype at each time point of CHX treatment. Wnd degradation after CHX treatment did not differ between wild-type and dFmr1 mutants.

(F) Representative immunoblot from wild-type and dFmr1 mutants. INPUT (left side of blot): 10% of the lysate prior to immunoprecipitation was collected for input controls, and blotting for dFMRP confirms the absence of dFMRP protein in dFmr1 mutants. IP (right side of the blot): dFMRP protein was immunoprecipitated from Drosophila heads, and as expected, no dFMRP was detected from dFmr1 mutants.

(G) RT-PCR products from input and immunoprecipitated samples from wild-type and dFmr1 flies. All mRNAs probed (listed on left of image) were present in the RNA input samples extracted from both genotypes. Futsch mRNA was used as a positive control and immunoprecipitated with dFMRP. rp49 mRNA was used as a negative control and did not immunoprecipitate with dFMRP. wnd mRNA immunoprecipitated with dFMRP in wild-type flies. No wnd mRNA is detected in dFmr1 mutant immunoprecipitation samples.

(H) qPCR quantification of wnd mRNA product detected from immunoprecipitated samples, normalized to wnd mRNA from input, and presented as relative mRNA levels compared to wild-type immunoprecipitated wnd mRNA. For dFmr1 mutants, all CT values were >38 or undetectable. All undetectable CT values were set to 40 for the purposes of quantification.

Statistical tests and exact p values reported in Table S1. For all quantifications: *p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001; NS, not significant; p > 0.05.