Fig 1: Suppressive effects of E3 ubiquitin ligases on neuronal TDP-43 aggregate formation. (A) Schematic presentation of the experiments for adenoviral TDP-43 aggregate formation. The differentiated 1464R cells were infected with adenoviruses expressing DsRed- and FLAG-tagged human wild-type (WT; AxDsRhTDP43WTFL) and C-terminal fragment (CTF; AxDsRhTDP43CTFFL) TDP-43 and EGFP-tagged human genes of interest (GOIs; AxhGOIEGFPs, i.e. AxhHSF1EGFP, AxhPJA1EGFP, AxhPJA1?REGFP, AxhPRKNEGFP, AxhRNF112EGFP or AxhRNF220EGFP), followed by incubation with 0.5 µm MG-132. (B) Western blot analysis of suppressive effects of adenoviral HSF1 and E3 ubiquitin ligases on phosphorylation and aggregate formation of adenoviral TDP-43. (C) Densitometric analysis of the Western blot data of RIPA-insoluble phosphorylated and total CTF TDP-43 (arrowheads in B; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP-treated control samples in the presence of MG-132. Results are presented as mean ± SD. Statistical comparison was performed by a two-tailed unpaired t-test (*P < 0.05). (D) The co-immunoprecipitation (Co-IP) assay showing that PJA1 (1), Parkin (2), RNF112 (3) and RNF220 (4) all bind to WT and CTF TDP-43 that are ubiquitinated. (E) Fluorescence microscopy of TuJ1-immunoreactive neurons infected with adenoviruses expressing DsRed-tagged human WT and CTF TDP-43 (AxhDsRTDP43WT + CTFFL) and EGFP-tagged human HSF1, PJA1, PJA1?R, Parkin (PRKN), RNF112 and RNF220 in the presence of MG-132. The nucleus was counterstained with Hoechst 33342.
Fig 2: Schematic diagram of recombinant adenovirus vectors encoding 3'-EGFP-tagged gene of interest (GOI), i.e. human wild-type (WT) HSF1 (AxhHSF1EGFP), WT and RING finger domain-lacking (?R) PJA1 (AxhPJA1EGFP, AxhPJA1?REGFP), WT Parkin (AxhPRKNEGFP), WT RNF112 (AxhRNF112EGFP) and WT RNF220 (AxhRNF220EGFP) (A), and 5'-DsRed (DsR)- and 3'-FLAG (FL)-tagged GOI, i.e. human WT and C-terminal fragment (CTF) TDP-43 (AxDsRhTDP43WTFL, AxDsRhTDP43CTFFL), WT and P525L FUS (AxDsRhFUSWTFL, AxDsRhFUSP525LFL), WT and G93A SOD1 (AxDsRhSOD1WTFL, AxDsRhSOD1G93AFL), WT and A53T a-synuclein (AxDsRhSNCAWTFL, AxDsRhSNCAA53TFL), ataxin-3 Q28 and Q84 (AxDsRhATXN3Q28FL, AxDsRATXN3Q84FL), and huntingtin exon 1 Q23 and Q74 (AxDsRhHttEx1Q23FL, AxDsRhHttEx1Q74FL) (B), and shRNAs for rat negative control (NC) and rat PJA1 coupled with EGFP (AxshNC/EGFP, AxshPJA1/EGFP) (C). These vectors contain the adenovirus type-5 genome lacking the E1A, E1B (?aE1A&?E1B) and E3 (?E3) regions to prevent the virus replication, the cytomegalovirus early enhancer/chicken ß-actin (CAG), U1 or cytomegalovirus (CMV) promoter on the 5' end, and the rabbit ß-globin polyA sequence (pA) on the 3' end.
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