Figure 1. Interaction between TDP-43 and the VHL/CUL2 complex.

(a) Schematic illustration of coupling of ReCLIP and in vitro ubiquitination to identify UPR-linked binding partners with TDP-43. First, recombinant TDP-43-FLAG proteins were incubated with S100T cell lysates potentially containing all components necessary for ubiquitination from HEK293A cells, followed by further reaction with the disulfide cross-linker DSP. After immunoprecipitation using anti-FLAG affinity gel, disulfide-linked proteins were released under mild reducing conditions. The eluates were analyzed by mass spectrometry. (b) In vivo pull-down assay of lysates from HeLa cells that were transfected with wild type (WT) TDP-43-GFP, TDP-43-GFP mutant with defective NLS (mNLS), and FLAG-CUL2, immunoprecipitated with anti-FLAG (left) or anti-GFP (right) affinity beads, and analyzed by Western blotting using anti-GFP or anti-FLAG antibodies, respectively. (c) Interaction of TDP-43 with VHL. Similarly, HEK293A cell lysates transfected with HA-VHL were immunoprecipitated with anti-HA affinity beads and immunoblotted for endogenous CUL2, elongin B, elongin C, and TDP-43. (d) The interaction between overexpressed TDP-43 and VHL was also shown by immunodetection of HA-VHL with immunoprecipitated TDP-43-FLAG. Lactacystin promoted binding of HA-VHL and TDP-43-FLAG. (e–p) Confocal laser micrographs of HEK293A cells expressing TDP-43-EGFP (as green) and HA-VHL (a–g, h–j) or Myc-CUL2 (k–m, n–p) 48 h after transfection. DAPI was used for counterstaining (blue). Occasional cytosolic inclusion of overexpressed WT TDP-43 colocalized with VHL (h–j) or CUL2 (n–p). Scale bar, 50 μm.