Journal: Neuropathology
Article Title: Praja1 RING ‐finger E3 ubiquitin ligase suppresses neuronal cytoplasmic TDP ‐43 aggregate formation
doi: 10.1111/neup.12694
Figure Lengend Snippet: 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 PJA1ΔR, 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 hPJA1ΔR is consistently localized in both RIPA‐soluble and insoluble fractions (lanes 3, 5, 7, 8), and adenoviral hPJA1v1, but not hPJA1ΔR, decreases RIPA‐insoluble phosphorylated CTF TDP‐43 (lanes 5, 7) in a similar manner to the experiment shown in Figure . 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 hPJA1ΔR (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.
Article Snippet: The blotted membrane was blocked with 3% skim milk and incubated overnight with rabbit anti‐FLAG (#7425; Sigma), rabbit anti‐TDP‐43 C‐terminus (405‐414) (#TIP‐TDP09; Cosmo Bio, Tokyo, Japan), rabbit anti‐phosphorylated TDP‐43 (pSer409/S410) (#TIP‐PTD‐P02; Cosmo Bio), rabbit anti‐PJA1 (#17687‐1‐AP; ProteinTech Japan, Tokyo, Japan), rabbit anti‐Myc Tag (#2278; Cell Signaling Technology, Danvers, MA, USA), rabbit anti‐ubiquitin K48 (#05‐1307; Sigma), rabbit anti‐UBE2D2/3 (#HPA003921: Sigma), rabbit anti‐UBE2E3 (#15488‐1‐AP; ProteinTech), rabbit anti‐UBE2K (#11834‐3‐AP; ProteinTech), and mouse monoclonal anti‐GAPDH (#ab8245; Abcam, Cambridge, UK) antibodies at dilutions of 1:1000, followed by incubation with biotinylated anti‐rabbit or anti‐mouse IgG (Vector Laboratories, Burlingame, CA, USA; 1:1000) for 1 h and streptavidin‐alkaline phosphatase (#11089161001; Sigma; 1:1000) for 1 h. Reactions were visualized by color development using nitroblue tetrazolium chloride (NBT) and 5‐bromo‐4‐chloro‐3‐indolylphosphate p‐toluidine salt (BCIP) (#11681451001; Sigma).
Techniques: Binding Assay, Western Blot, Expressing, Infection, Co-Immunoprecipitation Assay, Immunoprecipitation, Fluorescence