Black fluotrac microplates

FP was used to quantify binding affinities of the HPyV LTA cNLSs for IMPαΔIBB proteins. Two‐fold dilutions of recombinant IMPαΔIBB proteins were titrated into black Fluotrac microplates (Greiner Bio‐One, Kremsmünster, Austria) and FITC‐cNLS peptides were added to each well before making up to a total volume of 200 μL with tris‐buffered saline. The gain adjustment was made using a well with no FITC‐NLS peptide. The CLARIOstar Plus (BMG Labtech, Mornington, Australia) plate reader measured FP values, with each assay repeated in triplicate. Binding curves to calculate K_d and B_max values were generated with the one site specific binding least square fit function of Prism 9 (GraphPad) software.

Synthetic FITC-tagged AAV 09YN Cap-BR peptide was incubated (2 nm) with two-fold serially diluted IMPα concentrations (starting concentration 4.5 µM) across 23 wells to a complete volume of 200 µl per well with GST Buffer A (50 mM Tris, 125 mM NaCl). Fluorescence polarization (FP) measurements were recorded using a CLARIOstar Plus plate reader (BMG Labtech, Germany) in 96-well black Fluotrac microplates (Greiner Bio-One, Austria). Assays were performed as three independent experiments, each containing a negative control (no importin binding partner). Data from the three independent experiments was used to generate a binding curve using GraphPad Prism (Prism 9, Version 9.3.1).