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. 2023 Oct 23;14(10):695. doi: 10.1038/s41419-023-06222-z

Fig. 2. TRIM26 induces the K48-K63-linked polyubiquitination transition of GPX4.

Fig. 2

a HEK293T cells were transfected with Myc-GPX4 and the indicated ubiquitin mutant together with Flag-TRIM26 or not and then treated with MG132 (20 µM) for 6 h. Cell lysates were subjected to d-IP and IB with the indicated antibodies. b, c HEK293T cells transfected with the indicated plasmids were subjected to denaturing-IP (d-IP) and IB with the indicated antibodies. d HEK293T cells were transfected with Myc-GPX4 and indicated HA-Ub together with increasing amounts of Flag-TRIM26 and then treated with MG132 (20 µM) for 6 h. Cell lysates were subjected to d-IP and IB with the indicated antibodies. e HEK293T cells were transfected with Myc-GPX4 and indicated HA-Ub together with Flag-TRIM26 WT, Flag-TRIM26 CA or Flag-TRIM26 ΔR and then treated with MG132 (20 µM) for 6 h. Cell lysates were subjected to d-IP and IB with the indicated antibodies. f In vitro ubiquitination assay using the indicated purified proteins in the presence of E1, E2, and ATP. The reaction mixtures were incubated with Ni-NTA beads and then subjected to IB with an anti-GPX4 antibody. g–i HEK293T cells were transfected with Flag-TRIM26 and HA-Ub-K63 together with Myc-GPX4 WT or its mutants and then treated with MG132 (20 µM) for 6 h. Cell lysates were subjected to d-IP and IB with the indicated antibodies.