S200 instrument

SPR competition assays were performed
using a Biacore S200 instrument and the A–B–A inject
function. Recombinant USP21 was immobilized as described before for
the K_d measurements using the CM5 chip
experiments. The A–B–A injection method was used with
each of the SMOLs at 1 μM concentration with 30 s contact time
of analyte A (SMOLs) followed by ubiquitin titration that was run
in a 1:2 dilution series at a start concentration of 25 μM and
eight titration steps at a flow rate of 30 μL/min, with an association
time of 120 s and a dissociation time of 200 s at 15 °C. Each
titration was performed four times, and measurements were analyzed
using Biacore Insight Evaluation Software on double reference subtracted
sensorgrams.

Surface plasmon resonance
(SPR) experiments were performed using a Biacore S200 instrument at
25 °C. Samples were immobilized on a CM5 chip using amine-coupling
method according to the manufacturer’s instructions using a
running buffer containing 50 mM HEPES, 150 mM NaCl, 2 mM MgCl₂, and 0.05% Tween-20, pH 7.5. The binding affinity assay was
run with 2% DMSO in the running buffer at a flow rate of 30 μL/min
with a 60 s association and 200 s dissociation times. All of the assays
were performed twice, and binding affinity was calculated using BIAevaluation
software.