Fig. 4: Recruitment to PML enhances ubiquitylation of TDP-43.
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. As shown in Fig. 3d, but His-Ub was transfected together with FRB–PML–Myc and HA–TDP-43–FKBP. b, Enrichment of ubiquitylated HA–TDP-43–FKBP by Ni-NTA pull down in HEK293T cells expressing either WT or mutant (C57, 60S, L73E) FRB–PML–Myc together with HA–TDP-43–FKLBP and His-Ub. Signal intensities of high-molecular-weight bands (running above unmodified HA–TDP-43–FKBP) in pull-down samples (anti-HA) were quantified and normalized to the respective input intensities. Relative values are listed. c, 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 for 48 h and mock-treated or treated with TAK-981 (500 nM, 4 h). Rapamycin was added where indicated (100 nM, 4 h). Quantification was done as described in b. d, Enrichment of SUMO/Ub comodified HA–TDP-43–FKBP by HA-IP under denaturing conditions followed by Ni-NTA-pull down after HS (43 °C, 1 h). HEK293T cells were transfected with FRB–PML–Myc and HA–TDP-43–FKBP together with either His-Ub or an empty vector (e.v.). Rapamycin was added where indicated (100 nM, 4 h). HA–TDP-43–FKBP was enriched by HA-IP followed by denaturing Ni-NTA pull down of precipitated proteins. e, HEK293T cells were transfected with FRB–PML–Myc and HA–TDP-43–FKBP and treated with 100 nM rapamycin or DMSO for 4 h. TDP-43–FKBP was enriched by a denaturing HA-IP, and the ubiquitylation of TDP-43–FKBP was monitored with a pan-Ub or Ub-chain-specific (K48, K63) antibodies. f, Enrichment of ubiquitylated HA–TDP-43–FKBP by Ni-NTA pull down. HEK293T cells depleted of RNF4, Topors or both by siRNA or transfected with a nontargeting control siRNA followed by transfection with His-Ub, FRB–PML–Myc and HA–TDP-43–FKBP. Before lysis, cells were treated with rapamycin (100 nM, 4 h) or DMSO. Quantification was done as described in b. g, Enrichment of SUMOylated HA–TDP-43–FKBP by Ni-NTA pull down. As in f, but His-SUMO2 was transfected instead of His-Ub. Quantification was done as described in b.
