Biochemical characterization of ΔNLS-Tardbp fish. A Schematic drawing shows splicing products from zebrafish 2 orthologs of human TARDBP, tardbp and tardbpl. Zebrafish Tardbp has 2 RNA recognition motifs (RRM1 and RRM2) and a low-complexity domain (LCD). Tardbpl and Tardbpl_tv1 are splicing products from the tardbpl gene. While zebrafish Tardbpl does not have a low-complexity domain, zebrafish Tardbpl_tv1 has 2 RNA recognition motifs (RRM1 and RRM2) and a low-complexity domain. B Western blot analysis with the Tardbpl_tv1 specific antibody 16C8 detects Tardbpl_tv1 levels to be upregulated in tardbp ΔNLS/ΔNLS and tardbp -/- embryos compared to wildtype embryos. TDP DKO larvae in lane 4 serve as a control for the specificity of the Tardbpl_tv1 antibody. Asterics mark unspecific bands. α-tubulin serves as a loading control. C Schematic drawing highlights bipartite nuclear localization sequence (NLS) in zebrafish Tardbp. The ΔNLS-Tardbp mutation was generated in NLS1 by mutating the amino acids KRK to AAA. D Western blot analysis of 3 biological replicates with the Tardbp antibody 4A12 detects reduced Tardbp levels and a band shift to a higher molecular weight in CytoTDP embryos compared to Control (tardbp + / + ; tardbpl -/-) embryos. Semi-quantitative analysis of the Western blots revealed that the Tardbp levels normalized to α-tubulin in 5 dpf CytoTDP embryos (n = 3) are only 18% compared to Control (n = 3) (***p = 0.0004, Unpaired T test)
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