Fig. 2: The K63-linked polyubiquitin of GD-NT is regulated by TRAF1-OTUB1 axis.

A, B Interaction between GD-NT and E3 ligases. HEK293T cells were transfected with Flag-GD-NT and different HA-E3. 24 hours later, the cells were harvested for Flag-IP assay (A) and HA-IP assay (B). The preciptants were subjected to immunoblot with the indicated antibodies. C, D Deubiquitylation of GD-NT by DUBs. HEK293T cells were transfected with Flag-GD-NT, His-Ub-K63 only, and HA-TRAF/2 (C) or shTRAF1 (D). 24 hours later, the cells were harvested for Ni-NTA pull-down assays. The precipitants and WCL were immunoblotted with the indicated antibodies. E GD-NT interaction with DUBs. HEK293T cells were transfected with HA-GD-NT and different Flag-DUB. 24 hours later, the cells were harvested for IP assay. The preciptants were subjected to immunoblot with the indicated antibodies. F Deubiquitylation of GD-NT by DUBs. HEK293T cells were transfected with HA-GD-NT, His-Ub-WT, and Flag-OTUB1 or Flag-OTUB2. 24 hours later, the cells were harvested for Ni-NTA pull-down assays. The precipitants and WCL were immunoblotted with the indicated antibodies. G HEK293T cells were transfected with HA-GD-NT, His-Ub-K63 only, and Flag-OTUB1 or Flag-OTUB2. 24 hours later, the cells were harvested for Ni-NTA pull-down assays. The precipitants and WCL were immunoblotted with the indicated antibodies. H Similar to (G), except that shOTUB1 was used to knock down the expression of OTUB1. I TRAF1 and TRAF2 enhance the cytolytic activity of GD-NT. HEK293 cells were transfected with 3xFlag-GD-NT with or without HA-TRAF1 and HA-TRAF2. 48 hours later, morphological changes were observed using phase-contrast imaging (lower panel). Dying cells were stained with PI and photographed using fluorescent microscope (upper panel). J Similar to (I), except that the cells were subjected to LDH-based Cytotoxicity Assay. K Similar to (I), except that the WCL were immunoblotted with the indicated antibodies.