Fig. 2: SUMO2-modified TDP-43 is associated with PML NBs.
From: Induced proximity to PML protects TDP-43 from aggregation via SUMO–ubiquitin networks
a, Volcano plot displaying the results of IP-MS in HEK293T cells expressing Flag-4×SUMO2–TDP-43 or Flag-TDP-43. The difference in the log2 mean LFQ intensities between IPs from Flag-4×SUMO2–TDP-43 and Flag-TDP-43 expressing cells was plotted against the negative logarithmized P values of a two-sided Student’s t test (n = 3). Proteins significantly enriched in IPs from cells expressing Flag-4×SUMO2–TDP-43 (log2(4×SUMO2–TDP-43/TDP-43) > 1 and P < 0.05) are highlighted in red. TDP-43 and proteins associated with PML NBs are indicated. b, GOCC enrichment analysis of proteins interacting with Flag-4×SUMO2–TDP-43. Proteins that were exclusively identified in all three replicates of IPs from Flag-4×SUMO2–TDP-43 expressing cells and proteins that were significantly enriched in IPs from Flag-4×SUMO2–TDP-43 expressing cells (versus Flag-TDP-43) were combined into a dataset and used for GOCC enrichment analysis. Greater than fivefold enriched GOCC terms are shown. c, STRING cluster of proteins that were significantly enriched in a and are associated with the GOCC term ‘PML body’. d, HEK293T cells were transfected with Flag-4×SUMO2–TDP-43, Flag-SUMO2–TDP-43, Flag-TDP-43 or an empty vector. Flag-IP was performed to enrich TDP-43 variants and interacting proteins. Coprecipitated PML, Daxx and RNF4 were detected. e, Endogenous TDP-43 was immunoprecipitated from HEK293T cell lysates, and coprecipitation of PML was detected. Two major isoforms of PML are detected. f, HEK293T cells were transfected with Myc-tagged PML (WT or SIMmut) in combination with Flag-TDP-43 (WT, 1×SUMO, 4×SUMO) or an empty vector. TDP-43 was enriched by Flag-IP, and co-immunoprecipitated PML variants were detected. g, Enrichment of ubiquitylated Flag-4×SUMO2–TDP-43 by Ni-NTA pull down. HEK293T cells were depleted of RNF4 and Topors by siRNA. Subsequently, cells were transfected with His-Ub and Flag-4×SUMO2–TDP-43 and exposed to HS (43 °C, 1 h) or left untreated. Ubiquitylated proteins were enriched by denaturing Ni-NTA pull down. Signal intensities of high-molecular-weight bands (running above the height of unmodified Flag-4×SUMO2–TDP-43) in pull-down samples (anti-Flag) were quantified and normalized to the matching input intensities.
