Extended Data Fig. 5: Recruitment to PML enhances SUMOylation and ubiquitylation of disease associated TDP-43 mutants.
From: Induced proximity to PML protects TDP-43 from aggregation via SUMO–ubiquitin networks
a, Enrichment of ubiquitylated HA–TDP-43–FKBP by Ni-NTA pull down. HEK293T cells were transfected with His-Ub together with FRB–PML–Myc and HA–TDP-43–FKBP. Cells were treated with heat stress (43 °C, 1 h) or left untreated. Where indicated, cells were treated with rapamycin (100 nM, 4 h) and TAK-981 (500 nM, 4 h). Signal intensities of high-molecular-weight bands (running above the height of unmodified HA–TDP-43–FKBP) in pull-down samples (anti-HA) were quantified and normalized to the respective intensities in the matching input lane. Relative values are listed below the top right panel. b, Enrichment of SUMOylated HA–TDP-43–FKBP by Ni-NTA pull down. HEK293T cells were transfected with His-SUMO2 together with FRB–PML–Myc and HA–TDP-43–FKBP. Where indicated, cells were treated with rapamycin (100 nM, 4 h) and TAK-243 (10 µm, 4 h). Relative intensities of high-molecular-weight TDP-43–FKBP were determined as described in a. c, Enrichment of SUMOylated HA–TDP-43–FKBP by Ni-NTA pull down. HEK293T cells were transfected with His-SUMO2 together with FRB–PML–Myc and HA–TDP-43–FKBP (WT, P112H or K263E). Where indicated, cells were treated with rapamycin (100 nM, 4 h). d, Enrichment of ubiquitylated HA–TDP-43–FKBP by Ni-NTA pull down. HEK293T cells were transfected with His-Ub together with FRB–PML–Myc and HA–TDP-43–FKBP (WT, P112H or K263E). Where indicated, cells were treated with rapamycin (100 nM, 4 h).
