Fig. 5: TRIB3 stabilizes the FOXO1 protein by suppressing its ubiquitination. | Nature Communications

Fig. 5: TRIB3 stabilizes the FOXO1 protein by suppressing its ubiquitination.

From: TRIB3 supports breast cancer stemness by suppressing FOXO1 degradation and enhancing SOX2 transcription

Fig. 5

a, b HMLE cells transfected with a TRIB3-expressing vector vs. a control vector (a) or MCF7 cells transfected with TRIB3-shRNA vs. CTRL-shRNA (b) were incubated with 20 μM cycloheximide (CHX) for the indicated times. c, d The effect of TRIB3 depletion on FOXO1 degradation in vitro. MCF7 cells (c) were incubated with CHX (20 μM), CHX plus bafilomycin (BAF, 200 nM), or MG132 (10 μM) for the indicated times. Control or TRIB3-silenced MCF7 cells (d, top) and MDA-MB-231 cells (d, bottom) were incubated with DMSO, 200 nM BAF, or 10 μM MG132 for 8 h. The indicated proteins were detected by immunoblotting. e, f FOXO1 activity in HMLE cells (e) transfected with a control vector or a TRIB3-expressing vector, or in MCF7 cells (f) transfected with CTRL-shRNA or TRIB3-shRNA and either a control vector or a FOXO1-expressing vector. g, h The effect of TRIB3 on FOXO1 ubiquitination. HMLE (g) or MCF7 cells (h) were transfected with the indicated plasmid. His-ubiquitin-conjugated proteins were pulled down from cell lysates. Total protein lysates and Ni-NTA-agarose eluates were immunoblotted for FLAG, His, and TRIB3. Source data are provided as a Source Data file.

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