Fig 1: PERK assay development and validation.A) PERK titration curve. Graph represents PERK concentration in a two-fold serial dilution against PERK activity expressed as relative light units (RLU), fitted with nonlinear regression curve (Four Parameters Logistic Regression (4PL)). Data plotted as mean ± SEM from 3 independent experiments. B) Testing of different protein substrates. PERK natural substrates, eIF2a and NRF2, and SMAD3 were tested as protein substrates, in absence or presence of PERK. PERK activity was measured as a luminescence signal and expressed as RLU. Data are mean ± SEM from 3 independent experiments. Statistical analysis was 2-way ANOVA with Tukey’s post hoc test. **p < 0.01. C) Performance comparison of tested protein substrates. Graph show the gain of luminescence related to PERK autophosphorylation (no substrate protein). Data are mean ± SEM from 3 independent experiments. Statistical analysis was 1-way ANOVA with Dunnett’s post hoc test. *p < 0.05 and **p < 0.01. D) Testing an inhibitor and an activator. Effects of calcineurin B and GSK2606414 on PERK activity at different reaction times. Measured RLUs were normalized to basal PERK activity (control condition), which is set as 100% (dashed line). Data are mean ± SEM from 3 independent experiments. Statistical analysis was multiple t-test between control condition and GSK2606414 or calcineurin-B condition, respectively. *p < 0.05 and ***p < 0.001.