FIGURE 2.

Effect of altering the level or activity of ubiquitin-specific protease 14 (USP14) on steady-state levels of TARDBP, microtubule associate protein tau (MAPT), and prion protein (PRNP) proteins in mice. (a) Quantitation and (b) representative immunoblot of the steady-state levels of TARDBP in the hippocampi of 4- to 6-week-old wild type (wt), USP14-deficient (ax^J), neuronally over-expressed USP14 (TgUSP14), and neuronally over-expressed, catalytically-inactive USP14 (TgUSP14CA) mice. (c) Quantitation and (d) representative immunoblot of the levels of PRNP in the hippocampi of 4- to 6-week-old wt, ax^J, TgUSP14, TgUSP14CA mice. (e) Quantitation and (f) representative immunoblot of the levels of MAPT in the hippocampi of 4- to 6-week-old wt, ax^J, TgUSP14, TgUSP14CA mice. For TARDBP analysis, n = 7 ax^J, 4 TgUSP14, and eight TgUSP14CA extracts. For PRNP analysis, n = 8 ax^J, 6 TgUSP14, and 10 TgUSP14CA extracts. For MAPT analysis, n = 6 ax^J, 4 TgUSP14, and 8 TgUSP14CA extracts. An equivalent number of wild type extracts were analyzed for each protein. All quantitations are shown relative to wt control levels, and β-Tubulin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Data are shown as mean ± SEM. ****p < .0001. (g) Quantitative PCR depicting the levels of Tardbp, Prnp, and Mapt transcripts in the hippocampi of 5-week-old TgUSP14CA mice relative to wild type controls. n = 3. (h) Representative immunblot of HA-VME-Ub labeling assay of proteasomes from brains of wt, ax^J, TgUSP14, and TgUSP14CA mice. Blots were probed with an anti-HA antibody to identify catalytically-active USP14 and an anti-USP14 antibody to measure total USP14 levels. (i) Expression patterns of PRNP, TARDBP, and MAPT in hippocampal sections from 6-week-old wt, TgUSP14, ax^J, and TgUSP14CA mice. CALB2 (Calbindin 2) and tubulin beta chain (TUBB) were included as controls