Figure 7.
The endogenous association of EGFP–hnRNP K with targeted RNAs is unaffected by mutation of the JNK site. A, Experimental design of RNA-binding protein/RNA coimmunoprecipitation to test RNA association in vivo of EGFP fusion proteins (EGFP–hnRNP K, S189A, S189D). After expression of EGFP (control) and EGFP fusions, lysates were incubated with anti-GFP-coated beads to coimmunoprecipitate proteins with bound RNAs. In qRT-PCR, specifically bound RNAs reached threshold before RNAs that were nonspecific and therefore had smaller ΔCT values. B, Real-time qRT-PCR of RNAs eluted from anti-GFP immunoprecipitation, normalized to total input control. ΔCT values of NF-M and tau (hnRNP K targets) were significantly lower for EGFP–hnRNP K, S189A, and S189D compared with that of EGFP control (*p < 0.05, one-way ANOVA with Tukey's post hoc test) but not for peripherin (a nontarget), indicating specific binding. One-way ANOVA further showed no significant (ns) difference in binding of NF-M and tau among EGFP fusion proteins. Error bars indicate SD, n = 3 replicates, 30 embryos per group.