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Figure 1 | Molecular Neurodegeneration

Figure 1

From: Ubiquitin-specific protease 14 regulates c-Jun N-terminal kinase signaling at the neuromuscular junction

Figure 1

Transgenic expression of USP14CA in the nervous system. (A) Full length Usp14 cDNA containing a mutation of the active site cysteine to alanine (C114A) was cloned behind the Thy1.2 promoter. (B) Representative immunoblot of spinal cord extracts taken from 4- to 6- week-old wild type and ax J mice (± TgUsp14CA) mice using USP14-specific antisera. β-tubulin was included as a loading control. (C) Abundance of wild type Usp14 and transgenic Usp14CA transcripts obtained by RNA transcriptome analysis of brain tissue taken from 4- to 6-week-old wild type and TgUsp14CA mice. (D) Representative immunoblots of USP14 in whole brain lysates and brain proteasome fractions from 4- to 6-week-old wild type, TgUsp14CA, and TgUsp14 mice. β-tubulin was included as a loading control for whole brain and RPT1 was a loading control for the proteasome fraction. TgUsp14 mice express wild type USP14 behind the Thy1.2 promoter. (E) Ubiquitin vinyl methyl ester (Ub-VME) assay for DUB activity in proteasome fractions from the brains of 4- to 6-week-old wild type, TgUsp14, and TgUsp14CA mice. Ubiquitin is covalently attached to the active site cysteine of USP14, resulting in a slower migrating USP14 species denoted as USP14-Ub-VME. The mutant USP14CA cannot be labeled by Ub-VME. (F) Relative mRNA quantity of proteasome subunits and associated factors determined by qPCR, normalized to wild type (represented by dotted line on graph), from spinal cords of 6-week-old wild type and TgUsp14CA mice. n = 3 mice per genotype, run in triplicate. Data are shown as mean ± SEM (G) Assay of trypsin-like activity of proteasomes isolated from 4- to 6-week-old wild type and TgUsp14CA mice using the fluorogenic Boc-LRR-AMC substrate. n = 3 mice per genotype, and data are shown as mean ± SEM. See also Additional file 1.

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