Figure 4

TRIM25 catalyzes K11-linked polyubiquitination of HIF-1α at Lys719 and Lys721.A, IB of HIF-1α ubiquitination. HEK293T cells were transfected with Myc-HIF-1α, His-Ub-WT together with Flag-empty vector, Flag-TRIM25-WT or Flag-TRIM25-ΔRING expression plasmids for 20 h, then treated with MG132 (20 μM) for 4 h. B, IB of HIF-1α ubiquitination. HEK293T cells were transfected with Myc-HIF-1α, His-Ub-WT together with Flag-empty vector, Flag-TRIM25-WT or Flag-TRIM25-C/S (C50/53S) expression plasmids for 20 h, then treated with MG132 (20 μM) for 4 h. C, IB of endogenous HIF-1α ubiquitination in wild-type (TRIM25^+/+) and TRIM25-deficient (TRIM25^−/−) U251 cells treated with MG132 (20 μM) under hypoxia (1% O₂) for 4 h. D, IB of HIF-1α ubiquitination. HEK293T cells were transfected with Myc-HIF-1α, Flag-empty vector or Flag-TRIM25 together with His-ubiquitin mutants (K6R, K11R, K27R, K29R, K33R, K48R, and K63R) expression plasmids for 20 h, then treated with MG132 (20 μM) for 4 h. E, IB of HIF-1α ubiquitination. HEK293T cells were transfected with Myc-HIF-1α, Flag-empty vector or Flag-TRIM25 together with His-Ub-K11O or K11R expression plasmids for 20 h, then, treated with MG132 (20 μM) for 4 h. F, IB of the indicated proteins in HEK293T cells transfected with Myc-HIF-1α-WT or its mutant Myc-HIF-1α-K719/721R together with Flag-empty vector or Flag-TRIM25 expression plasmids. G, IB of ubiquitination of HIF-1α and its mutant HIF-1α-K719/721R. HEK293T cells were transfected with His-Ub-WT, Myc-HIF-1α-WT or Myc-HIF-1α-K719/721R together with Flag-empty vector or Flag-TRIM25 expression plasmids for 20 h, then, treated with MG132 (20 μM) for 4 h. H, IB of ubiquitination of HIF-1α and its mutant HIF-1α-K719/721R. HEK293T cells were transfected with His-Ub-K11O, Myc-HIF-1α-WT or Myc-HIF-1α-K719/721R together with Flag-empty vector or Flag-TRIM25 expression plasmids for 20 h, then treated with MG132 (20 μM) for 4 h.