Quantitative assessment of protein–RNA interactions by the dual fluorescence RNA-binding assay. (A) Schematic representation of the dual fluorescence RNA-binding assay using conventional or PAR UV cross-linking protocols. (B,C) eGFP and MOV10–YFP cell lines were induced for 16 h and irradiated with 254-nm UV light. After lysis, control samples were treated with RNases. Recombinant proteins were immunoprecipitated using GFP-multiTrap. Co-immunoprecipitated RNAs were detected by hybridization with either 40 nM oligo(DT)25, oligo(DT)11, or random hexamers. The graphs represent relative green (eGFP/YFP) and red (Tred) fluorescence levels (B) and their ratios normalized to RNase-treated samples (C). (D,E) Dual fluorescence RNA-binding assay of tetracycline (Tet+) induced and noninduced (Tet−) Tet-on cell lines expressing eGFP or MOV10–YFP. Lysates were mock, RNase- or DNase-treated prior to immunoprecipitation in GFP-multiTrap. The graphs represent relative green (eGFP/YFP) and red (Tred) fluorescence levels (D), and their ratios normalized to RNase-treated samples (E). (F,G) As in D and E, but immunoprecipitation was carried out in microtubes using GFP-Trap_A. The graphs represent relative green (eGFP/YFP) and red (Tred) fluorescence levels (F) and their ratios normalized to RNase-treated samples (G). All graphs were generated from two biological and three technical replicates (total of six immunoprecipitations). (**) P < 0.01; (***) P < 0.001. Horizontal red and green lines represent the averaged Tred or eGFP background signals, respectively, extracted from control samples.
