Softworx acquisition and deconvolution software

For all SPR experiments, kinetic parameters were determined by curve-fitting using TraceDrawer software (Ridgeview Instruments AB) fit with a one-to-one model. Statistical analyses (mean ± SEM; 4–6 replicates) of SPR data were completed using Microsoft excel. For ITC experiments, data were analyzed with a one-site binding model assuming a binding stoichiometry of 1:1 using NITPIC,^{49 (link)} SEDPHAT^{50 (link)} and GUSSI.^{50 (link)} Statistical analyses (mean ± SEM; 2–4 replicates) of ITC data were completed using Microsoft excel. For fluorescent imaging, images were processes by SoftWorX acquisition and deconvolution software (GE Healthcare). All images are single, deconvolved optical sections. For fluorescent F2H screens, 120 cells were scored for the accumulation of GFP and/or GFP-RRP1B variants at the gene locus and the percentage reported. Graphs report plot line profiles for red and green fluorescence over 2 μm at the gene locus. For IP and Western Blots, Chemiluminescent signals were captured using a ChemiDoc MP Imaging System (BioRad) and band intensities quantified using Image Lab software (BioRad).

For live imaging, cells were cultured in 35 μm optically clear polymer-bottom μ-dishes (ibidi) and transiently transfected for 18 hrs using PEI. Growth medium was replaced with Phenol Red-free CO₂ independent medium (ThermoFisher) and DNA stained by incubating the cells for 20 min at 37°C in medium containing 0.25 μg/m Hoechst No. 33342 (Sigma-Aldrich). Images were acquired using a DeltaVision CORE widefield fluorescence system fitted with 40x NA 1.2 and 60× NA 1.4 PlanApochromat objectives (Olympus), CoolSNAP charge-coupled device (CCD) camera (Roper Scientific) and environmental chamber. The microscope was controlled and images processed by SoftWorX acquisition and deconvolution software (GE Healthcare). All images are single, deconvolved optical sections.