Figure 4

Evaluation of the bead halo assay system. (a–d) The fluorescence intensity of GST-mCherry-TrnSR and GFP-SRSF1 as measured for 27 single beads is shown to evaluate the deviation of assay values. (a) BG-subtracted mCherry, (b) BG-subtracted GFP, and (d) BG-subtracted GFP/mCherry values are plotted against the bead diameters. (c) BG-subtracted GFP intensities are plotted against the BG-subtracted mCherry intensities of the same beads. C.V., coefficient of variation of the fluorescent values; R, correlation coefficient between the fluorescent values and the bead diameters (a, b, d) or between the mCherry and GFP intensities (c). (e–j) The dosage of bacterial extract containing GFP-cargo was varied from zero to that used in the final assay to evaluate the dose–response. (e) GST-mCherry-TrnSR and GFP-PQBP1, (f) GST-mCherry-TrnSR and GFP-RTF2, (g) GST-mCherry-Imp13 and GFP-MAGOH, (h) GST-mCherry-Imp13 and GFP-CHRAC1, (i) GST-mCherry-Imp13 and GFP-PPIE, and (j) GST-mCherry-Imp13 and GFP-UBE2I were analyzed by BHA. Numbers are the 3rd-Z-ranks of the cargoes. Three images for one GFP-cargo dosage were quantified, and the mean intensities of the GFP without BG subtraction (light green), BG of GFP (black), and BG-subtracted GFP (green) divided by the mean BG-subtracted mCherry intensity are plotted with the S.D. indicated by the error bar. Note that the BG of mCherry is low because the beads were used after washing out the unbound GST-mCherry-NTR, while the BG of GFP rises with the dose of GFP-cargo because it includes the fluorescence of free GFP-cargo that is in equilibrium with bead-bound GFP-cargo. All the NTR used are WT. The fitted curves were drawn by Microsoft Excel.