Fluorescence polarization PP2A binding assays

Following the instructions of the manufacturer, 100 µM of FAM122A_ID(S120C) (or variants) or ARPP19(S10C) was labelled with Alexa Fluor 488 C5 Maleimide (ThermoFisher) using 1:10 protein to fluorophore ratio. The mixture was incubated for 2 h in the dark at room temperature at pH 7.0 and excess β-mercaptoethanol (1.2× the concentration of the fluorophore) was added to inactivate any unreacted Alexa Fluor 488. Labelled FAM122A_ID(S120C) (or variants) or ARPP19(S10C) was recovered by analytical SEC (Superdex 75 Increase 10/300 (Cytiva)) and used for the fluorescence polarization assays. The labelled FAM122A_ID(S120C) (or variants) or ARPP19(S10C) are hereafter referred to as FAM122A_ID-tracer, or ARPP19-tracer.

The fluorescence polarization assays were standardized using black 384-well low volume round bottom microplates (Corning, 4411) with 15 µl solution per well. The measurements were performed using a CLARIOstarPlus (BMG LABTECH Inc) microplate reader (using reader control software version 5.7 R2) set up to 482 ± 16 nm excitation, 530 ± 40 nm emission, and dichroic long pass filter 504 nm with reflection ranging between 380–497 nm and transmission ranging between 508–850 nm. For the dissociation constant (K_d) binding measurements, all dilutions were made into fluorescence polarization buffer (10 mM HEPES pH 7.0, 150 mM NaCl, 0.5 mM TCEP, 0.01% Triton X-100, 0.1 mg ml⁻¹ BSA). A predilution of FAM122A_ID-tracer/ARPP19-tracer was prepared for 0.3 nM and a serial dilution of PP2A:B55 was made at 3 times the final concentration. Five microlitres of FAM122A_ID-tracer/ARPP19-tracer, 5 µl of serially diluted PP2A:B55 complex and 5 µl of fluorescence polarization buffer were distributed into the 384-well microplate, resulting in a 0.1 nM final concentration of FAM122A_ID-tracer or ARPP19-tracer. All assay experiments were repeated in triplicate and incubated for 30 min in the dark and sealed at room temperature before reading. The experiments were independently repeated ≥3 times and the averaged K_d and s.d. values were reported. The data was evaluated using GraphPad Prism 9.5.