Figure 4. OTULIN variants compromise binding of OTULIN to linear ubiquitin and differentially affect OTULIN’s intrinsic stability and catalytic activity.

Biochemical characterization of recombinant OTULIN variants by means of differential scanning fluorimetry (DSF) (A) and surface Plasmon resonance (SPR) measurements (B).

1. DSF measurements with OTULIN^WT, OTULIN^M86I, or OTULIN^W167S. Melting temperatures (T _m ) are calculated from five independent experiments with standard deviations. *P = 0.011; ****P = 6.64 × 10⁻¹⁸, unpaired t‐test.
2. SPR measurements and steady‐state binding curves with calculated dissociation constants (K_d ) after injection of a concentration series of OTULIN^C129A, OTULIN^C129A/M86I, or OTULIN^C129A/W167S to CM5‐immobilized di‐ubiquitin chains.
3. Linear ubiquitin linkages isolated from A549 OTULIN KO cells by M1 TUBE assay were incubated with increasing concentrations of recombinant OTULIN^WT, OTULIN^M86I, and OTULIN^W167S or catalytically inactive OTULIN^C129A as control for 1 h. Afterward, samples were subjected to analysis by Western blot for the indicated proteins. Images are representative of three independent experiments.