Fig. 3: Recruitment to PML enhances SUMOylation of TDP-43.
From: Induced proximity to PML protects TDP-43 from aggregation via SUMO–ubiquitin networks
a, Model of the rapamycin-induced recruitment of PML to TDP-43. FKBP was fused to the C terminus of HA–TDP-43, and FRB was fused to the N terminus of PML–Myc. Upon addition of rapamycin, HA–TDP-43–FKBP is recruited into a ternary complex with FRB–PML–Myc and rapamycin. Panel a is created with BioRender.com. b, HA-IP was performed in HEK293T cells expressing FRB–PML–Myc and HA–TDP-43–FKBP for 48 h. Cells were mock-treated or treated with rapamycin (100 nM, 4 h). c, HeLa cells expressing endogenously tagged RNF4-3×Flag were transfected with FRB–PML–Myc and HA–TDP-43–FKBP for 24 h and either mock-treated or treated with rapamycin (100 nM, 3 h). Inset areas are indicated with yellow squares. Scale bar = 5 µm. Statistical analysis is provided in Extended Data Fig. 3a. d, 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 for 48 h and were treated with HS (43 °C, 1 h), SA (0.5 mM, 1 h) or left untreated. Rapamycin was added where indicated (100 nM, 4 h). SUMOylated proteins were enriched by denaturing Ni-NTA pull down. e, As in d, but cells were left untreated. HEK293T cells were transfected with FRB–PML–Myc (WT, C57,60S or L73E) together with HA–TDP-43–FKBP and His-SUMO2. 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. f, HeLa cells expressing TDP-43–FKBP–HA from the endogenous TDP-43 locus together with FRB–PML–Myc were treated with DMSO or 100 nM rapamycin for 4 h. TDP-43–FKBP–HA was enriched by HA-IP under denaturing conditions and immunoblotted for SUMO2/SUMO3 conjugates. g, U2OS WT and PML-KO cells were subjected to 1 h of HS at 43 °C or left untreated. SUMOylated proteins were enriched by IP. Rapa., rapamycin; KO, knockout.
