Fig. 4: MIB1 ubiquitylates and drives the proteasomal degradation of TOP3B independently of TDRD3 and TDRD3-USP9X complex works downstream of MIB1 to limit TOP3B degradation. | Nature Communications

Fig. 4: MIB1 ubiquitylates and drives the proteasomal degradation of TOP3B independently of TDRD3 and TDRD3-USP9X complex works downstream of MIB1 to limit TOP3B degradation.

From: The TDRD3-USP9X complex and MIB1 regulate TOP3B homeostasis and prevent deleterious TOP3B cleavage complexes

Fig. 4

a TOP3B pulldown-Western blot experiments showing TOP3B-MIB1 interaction in HEK293 cells. b MIB1 pulldown-Western blot experiments confirming TOP3B-MIB1 interaction in HEK293 cells. c TDRD3 pulldown-Western blot experiments showing TDRD3-MIB1 interaction in HEK293 cells. d TOP3B pulldown-Western blot experiments showing that TOP3B interacts with MIB1 both in wild type and siTDRD3-transfected HEK293 cells. e MIB1 depletion increases endogenous TOP3B protein level without affecting TDRD3 levels. f MIB1 ubiquitylates TOP3B independently of TDRD3. HEK293 cells were transfected with HA-Ubiquitin construct and siTDRD3, siMIB1 or both. Before harvest, cells were treated with MG132 (1 µM, 24 h) and subjected to TOP3B pulldown. Pulled down and input samples were resolved on SDS-PAGE and probed with Ubiquitin, TOP3B, TDRD3, and MIB1 antibodies (GAPDH as loading control). The figure is representative of three independent experiments. g Ectopic expression of MIB1 increases TOP3B ubiquitylation. HEK293 cells were transfected with HA-Ubiquitin construct and MIB1-FLAG construct. Before harvest, cells were treated with MG132 (1 µM, 24 h) as indicated and subjected to TOP3B pulldown. Pulled down and input samples were resolved on SDS-PAGE and probed with Ubiquitin, TOP3B, TDRD3, and MIB1 antibodies (GAPDH as loading control). The figure is representative of three independent experiments. h MIB1 depletion does not affect TDRD3 ubiquitylation. Cells were transfected with HA-Ubiquitin construct and siMIB1 as indicated for 48 h. Before harvest, cells were treated with MG132 (1 µM, 24 h) as indicated and subjected to TDRD3 pulldown. Pulled down and input samples were resolved on SDS-PAGE and probed with Ubiquitin, TDRD3 and MIB1 antibodies (GAPDH as loading control for input samples). The figure is representative of three independent experiments. i USP9X antagonizes MIB1 to stabilize TOP3B protein. HEK293 cells were transfected with the indicated siRNAs for 48 h. j Schematic representation of the homeostatic regulation of TOP3B protein levels by TDRD3-USP9X and MIB1. The TOP3B interacting E3 ligase MIB1 ubiquitylates and targets TOP3B for proteasomal degradation while the TDRD3-USP9X complex deubiquitylates and protects TOP3B from proteasomal degradation. Source data are provided as a Source Data file.

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