Fig. 5: Inhibitor-mediated engagement and activity of ERO1A. | Cell Death & Disease

Fig. 5: Inhibitor-mediated engagement and activity of ERO1A.

From: Small molecule-mediated inhibition of the oxidoreductase ERO1A restrains aggressive breast cancer by impairing VEGF and PD-L1 in the tumor microenvironment

Fig. 5

A Schematic representation of the recombinant mouse ERO1A construct, indicating the ULP (a Sumo protease) cleavage site. B Coomassie-stained SDS-PAGE representing ERO1A cleaved from the upstream GST_SUMO, GST_SUMO, and BSA as a protein loading control. C Coomassie-stained nonreducing SDS-PAGE of ERO1A (1 μM) reacted with 20 μM of the 23 compounds. Representative experiments reproduced twice with different batches of ERO1A protein. On the right, bar graphs indicating the reduced ERO1A on total ERO1A (reduced ERO1A + oxidized ERO1A); the DTT-treated ERO1A was arbitrarily set to 1 (mean ± SEM, One-Way ANOVA). D Coomassie-stained non-reducing SDS-PAGE indicating ERO1A exposed to DTT or the concentrations indicated of EN460, I2, I3 and I15. Representative experiment reproduced twice. On the right, bar graphs indicating the reduced ERO1A on total ERO1A (mean ± SEM, One-Way ANOVA). E Coomassie-stained SDS-PAGE representing ERO1A, PDI1A1, and BSA as a protein loading control. F Concentration-dependent inhibition of the ERO1A-dependent AUR fluorescence in a kinetic assay employing ERO1A and PDIA1 (raw data in Fig Suppl. 2B). Curves were fitted using the equation: “log(inhibitor) vs. response - Variable slope” in Prism 10, to obtain the IC50 values with corresponding 95% interval of confidence, which were: 6.2 µM (5.1–7.5), 3.5 µM (2.9–4.3) and 8.1 µM (6.7–9.9) for EN640, I3 and I2, respectively.

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