Figure 2
The ALS-mutations in the GR segment do not affect the binding to G4-RNA. (A) The structural features of GST fusion GR wild-type and mutant proteins and SPR sensorgrams of the interaction between G4-RNA and GST fusion proteins. Terminal biotinylated poly dT16 was bound to the streptavidin-coated sensor chip through streptavidin–biotin complex, to which poly-dA16 tailed G4-RNA (PSD-95 dA16 and CaMKIIα dA16, 20 nM) was immobilized by hybridization. The test protein (50 nM) was added to measure interaction with G4-RNAs. (B) UV cross-linking for detection of G4-RNA and binding protein complexes. The reaction mixtures were irradiated at 254 nm at 200 mJ/cm2 and analyzed by denaturing gel mobility shift assay. Shifting bands were enhanced in both fluorescent probes (Table S2) with increased GST-GR proteins (1, 2 or, 4 pmol each). Several minor bands are seen that may have been cleaved during electrophoresis, but only the major band was used for quantification (for original images, see Fig. S2). The gel was stained with Coomassie Brilliant Blue and the blocking agent BSA signals were used to correct the data as a loading control. The experiments were performed thrice, and the y-axis values represent the mean ± SEM at 4 pmol. The statistical significance was not confirmed by a two-tailed Student’s t-test (n.s.).
