Journal: Brain Communications

Article Title: Immunoproteasome deficiency results in age-dependent development of epilepsy

doi: 10.1093/braincomms/fcae017

Figure Lengend Snippet: Immunoproteasome expression in the brains of young and old mice. ( A ) A western blot analysis of immunoproteasome distribution in the spleen, thymus, liver and brain of 2-month-old female WT mice. A representative western blot is shown (left side). The bar graphs display a relative abundance of low - molecular-weight protein 2 (LMP2) and low-molecular-weight protein 7 (LMP7) normalized to the proteasome subunit α3 ( n = 3–4). The statistical analysis was performed by using one-way ANOVA. Data are presented as mean ± SD, * P = 0.01–0.05, *** P < 0.001. Uncropped blots are shown in . ( B ) A western blot of polyubiquitination in the hippocampi of 2-month-old and 1-year-old female WT mice. A representative western blot is shown (left side), and the bar graphs display a relative polyubiquitin signal normalized to β-actin ( n = 3). The statistical analysis was performed by using an unpaired t -test. Data are presented as mean ± SD, * P = 0.01–0.05. ( C ) A western blot of 2- and 8-month-old female WT and TKO mice displays an increase in the polyubiquitination of TKO hippocampi compared with WT hippocampi. A representative western blot is shown. The bar graphs display polyubiquitination levels normalized to β-actin ( n = 3–4). Statistical significance for ( B ) and ( C ) was analysed by using an unpaired t -test. Data are presented as mean ± SD, * P = 0.01–0.05. Uncropped blots are shown in . ( D) A fluorescence staining of LMP7 (green) and NeuN (magenta, a neuronal marker) in the brains of young and old WT mice. A staining of the brains of old TKO mice is shown as a negative control. ( E ) A fluorescence staining of phospho-tau (AT8 = green, white arrows) in the CA3 region of the brains of WT versus TKO mice. The neuronal nuclear protein (NeuN, magenta) serves as a neuronal marker, and DAPI (blue) stains the nuclei. ( F ) A diaminobenzidine staining of phospho-tau (AT8) in the hippocampi of old WT and TKO mice (CA1).

Article Snippet: The TKO mice were kindly provided by Kenneth Rock (University of Massachusetts Medical School) and Regeneron Pharmaceuticals.

Techniques: Expressing, Western Blot, Molecular Weight, Fluorescence, Staining, Marker, Negative Control

Journal: Brain Communications

Article Title: Immunoproteasome deficiency results in age-dependent development of epilepsy

doi: 10.1093/braincomms/fcae017

Figure Lengend Snippet: Immunoproteasome deficiency increases excitability and the risk of developing epilepsy. ( A ) TKO mice develop epileptic seizures at an age of 5–10 months. Female mice ( n = 141) were scored over 20 months. The graph displays the occurrence of seizures in per cent. Statistical significance was analysed by performing a Mantel–Cox test **** P < 0.0001. ( B–G ) Seizure susceptibility of female TKO mice was tested via an i.p. injection of KA (10 mg/kg). The mice were monitored for 60 min after injection. ( B ) A representative EEG of female WT, LMP7 KO and TKO mice. ( C ) The Racine score was estimated in intervals of 5 min. Statistical significance was analysed by using one-way ANOVA (Turkey’s multiple comparisons test, n = 7 mice/group): WT versus LMP7 KO n.s., WT versus TKO **** P < 0.0001, LMP7 KO versus TKO **** P < 0.0001. ( D ) The number of seizures per 5 min was calculated by counting the occurrence of seizures over 40 min ( n = 7 mice/group). Statistical significance was analysed by using one-way ANOVA: WT versus LMP7 n.s., LMP7 versus TKO n.s., WT versus TKO * P = 0.0122. ( E ) The graph displays the survival of animals ( n = 7 mice/group). Statistical significance was analysed by using the Mantel–Cox test: WT versus TKO **** P < 0.0001, WT versus LMP7 KO n.s., LMP7 KO versus TKO ** P = 0.0031. ( F ) The behavioural score of young female WT and TKO animals after an injection of KA ( n = 4 mice/group). Statistical significance was analysed by performing a paired t -test. *** P = 0.0001. ( G ) The Survival of young and old female TKO mice after an injection of KA ( n = 4–7 mice per group). Statistical significance was analysed by using the Mantel–Cox test ** P = 0.003.

Article Snippet: The TKO mice were kindly provided by Kenneth Rock (University of Massachusetts Medical School) and Regeneron Pharmaceuticals.

Techniques: Injection

Journal: Brain Communications

Article Title: Immunoproteasome deficiency results in age-dependent development of epilepsy

doi: 10.1093/braincomms/fcae017

Figure Lengend Snippet: TKO animals display several neurological disorders apart from recurrent seizures. ( A ) Female TKO animals show increased anxiety in the open field study compared with age-matched WT mice (>1-year-old mice; n = 5 mice/group). The statistical analysis was performed by using an unpaired t -test. ( B ) A gait analysis of >1-year-old female TKO and WT mice ( n = 5 mice/group). The stride length, stride width and toe spread were measured. The SD value of each mouse was calculated. ( C ) Calbidin staining in female WT and TKO mice shows a significant loss of Purkinje cells in TKO animals ( n = 4 mice/group). The number of Purkinje cells per 10 mm was calculated. The statistical analysis was performed by using the unpaired t -test. * P = 0.01–0.05; ** P = 0.001–0.01.

Article Snippet: The TKO mice were kindly provided by Kenneth Rock (University of Massachusetts Medical School) and Regeneron Pharmaceuticals.

Techniques: Staining

Journal: bioRxiv

Article Title: Immunoproteasomal Processing of Isolevuglandin Adducts in Hypertension

doi: 10.1101/2023.04.10.536054

Figure Lengend Snippet: Immunoproteasome triple knockout mice (TKO) and control C57BL/6 mice were treated with angiotensin II. Radiotelemetry was used to measure (A) systolic, (B) diastolic, and (C) mean blood pressure. Blood pressure was analyzed using 2-way ANOVA ( n =5-7). (D) Representative flow cytometry diagrams for CD4 + and CD8 + T cells harvested from Kidney. (E) Quantitation of CD8 + renal T cells. (F) Representation of CD8 + T cells as a percentage of total CD3 + T cells. (G) Quantitation of total CD3 + renal T cells. (H) Quantitation of FRET + DCs harvested from aorta. (I) Representation of FRET + aortic DCs as a percentage of total DCs. (J) Representative flow cytometry plots for FRET in DCs harvested from aorta. Data were analyzed by two-tailed Student’s T-test or Mann Whitney U Test ( n = 6-7).

Article Snippet: Immunoproteasome triple knockout mice (TKO) were obtained from Regeneron Therapeutics.

Techniques: Triple Knockout, Control, Flow Cytometry, Quantitation Assay, Two Tailed Test, MANN-WHITNEY