Fig 1: PJA1 binding to TDP-43 and E2 ubiquitin conjugating enzyme UBE2E3. (A) Western blot analysis of suppressive effects of adenovirus expressing human PJA1v1, but not PJA1?R, on phosphorylation and aggregate formation of TDP-43 after DsRed- and FLAG-tagged wild-type (WT) and CTF TDP-43 adenovirus infection in the presence of 0.5 µM MG-132 (lanes 3–8). The CTF TDP-43 adenovirus preferably induces RIPA-insoluble phosphorylated TDP-43 (lanes 4, 6). Adenovirally-induced hPJA1v1 and hPJA1?R is consistently localized in both RIPA-soluble and insoluble fractions (lanes 3, 5, 7, 8), and adenoviral hPJA1v1, but not hPJA1?R, decreases RIPA-insoluble phosphorylated CTF TDP-43 (lanes 5, 7) in a similar manner to the experiment shown in Figure 3F. Endogenous UBE2E3 is also detected both in RIPA-soluble and insoluble fractions. (B) The co-immunoprecipitation (Co-IP) assay shows that PJA1 preferentially binds to CTF TDP-43 rather than WT TDP-43 (PJA1 blot; lanes 3, 5, 7, 8), while CTF TDP-43 is consistently ubiquitinated irrespective of adenoviral PJA1 induction (Ubiquitin K48 blot; lanes 4–8). Both native and adenoviral UBE2E3 are co-immunoprecipitated with WT and CTF TDP-43 (UBE2E3 and Myc-Tag blots). (C) Co-IP assay indicates that TDP-43 and/or PJA1 bind to UBE2E3 but not UBE2D2/3 or UBE2K. (D) Fluorescence micrographs of TuJ1-immunoreactive differentiated neurons co-infected with DsRed-tagged WT and CTF TDP-43 adenoviruses and EGFP-tagged hPJA1v1 (top row) or hPJA1?R (bottom row) adenovirus in the presence of 0.5 µM MG-132. The nucleus is counterstained with Hoechst 33342. Two types of co-localization (i.e., perinuclear round fluorescence [arrows] and cytoplasmic amorphous aggregates [arrowheads]), are observed.