Journal: Scientific Reports

Article Title: Comparative synaptosome imaging: a semi-quantitative method to obtain copy numbers for synaptic and neuronal proteins

doi: 10.1038/s41598-018-33130-6

Figure Lengend Snippet: Numbers of primary antibodies per synaptosome for over 100 targets.

Article Snippet: Calcineurin , 11.718 , 0.188 (SEM) , 138.67 (isoform B) , 91.82 , Synaptic Systems , 387 002 , rabbit polyclonal , 1:1000.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: Calcineurin depletion coincides with phosphorylated TDP-43 deposition in a mouse model of ALS/FTLD-TDP

doi: 10.1186/s40478-025-02192-9

Figure Lengend Snippet: Reduced calcineurin in rNLS8 mouse brain at symptomatic timepoints. Representative images of calcineurin immunostaining in control animals, and rNLS8 animals at 1 week, 4 weeks, and 7 weeks post-induction. a – d hippocampus, h – k motor cortex, o – r dorsal striatum. Scale bars: 100 µm. Insets show higher magnification detail. Immunoreactivity quantification at 1 week, 4 weeks, and 7 weeks post-induction in hippocampus ( e – g ), motor cortex ( l – n ), and dorsal striatum ( s – u ). Statistical analysis is by unpaired t -test, two-tailed [ n = 8 control, 15 rNLS8 animals (1 week); 8 control, 16 rNLS8 animals (4 weeks); 15 control, 15 rNLS8 animals (7 weeks); ns: p > 0.05, *: p ≤ 0.05, and ****: p ≤ 0.0001]. All bar graphs represent mean ± SEM

Article Snippet: Sections were treated for endogenous peroxidases with 3% hydrogen peroxide in PBS (pH 7.4) for 30 min at room temperature, blocked in 5% non-fat milk in PBS for one hour at room temperature, and incubated with one of the following primary antibodies overnight at 4 °C: calcineurin (CN-A1 mAb, Rockland 200-301-P23, 1:500), NeuN (A60 mAb, MilliporeSigma MAB377, 1:2000) and pTDP-43 (S409/410, clone 1D3, gift from Dr. Manuela Neumann, 1:400) [ ].

Techniques: Immunostaining, Control, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Calcineurin depletion coincides with phosphorylated TDP-43 deposition in a mouse model of ALS/FTLD-TDP

doi: 10.1186/s40478-025-02192-9

Figure Lengend Snippet: Calcineurin and CDC7 protein level changes correspond to onset of TDP-43 phosphorylation in rNLS8 mouse brain. Representative immunoblots and densitometry analysis of chemiluminescence signals for total TDP-43, phosphorylated TDP-43 (pTDP-43), calcineurin, CDC7, and actin for control animals, and rNLS8 animals at 1 week, 4 weeks, and 7 weeks post-induction. Quantification is normalized to actin. a – e hippocampus, f – j cortex, and k – o striatum. Statistical analysis is by one-way ANOVA, followed by Tukey’s post-test ( n = 4–6 control animals and 4–6 rNLS8 animals for 1 week, 4 weeks, and 7 weeks post-transduction; ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, and ****: p ≤ 0.0001). All bar graphs represent mean ± SEM

Article Snippet: Sections were treated for endogenous peroxidases with 3% hydrogen peroxide in PBS (pH 7.4) for 30 min at room temperature, blocked in 5% non-fat milk in PBS for one hour at room temperature, and incubated with one of the following primary antibodies overnight at 4 °C: calcineurin (CN-A1 mAb, Rockland 200-301-P23, 1:500), NeuN (A60 mAb, MilliporeSigma MAB377, 1:2000) and pTDP-43 (S409/410, clone 1D3, gift from Dr. Manuela Neumann, 1:400) [ ].

Techniques: Phospho-proteomics, Western Blot, Control, Transduction

Journal: Acta Neuropathologica Communications

Article Title: Calcineurin depletion coincides with phosphorylated TDP-43 deposition in a mouse model of ALS/FTLD-TDP

doi: 10.1186/s40478-025-02192-9

Figure Lengend Snippet: Reduced calcineurin transcription in symptomatic rNLS8 mouse brain. a UMAP showing cluster distribution of single nucleus RNA sequencing(snRNAseq) combined from 5 control animals and 5 rNLS8 animals at 4 weeks post-induction. b Average expression of calcineurin heterodimer catalytic and regulatory genes (Ppp3ca, Ppp3cb, Ppp3cc, Ppp3r1, Ppp3r2) throughout UMAP space. c Log-normalized expression of the calcineurin catalytic subunit Ppp3ca in control (WT) and rNLS8 animals in cortical layer 6 intratelencephalic-projecting glutamatergic neurons (L6 IT), cortical layer 4/5 intratelencephalic-projecting glutamatergic neurons (L4/5 IT), cortical layer 2/3 intratelencephalic-projecting glutamatergic neurons (L2/3 IT), hippocampal CA1 glutamatergic neurons (CA1-Pros), cortical layer 6 corticothalamicprojecting glutamatergic neurons (L6 CT), and cerebellar granule glutamatergic neurons (CB Gran). Significance values are p adj values generated from cluster level pseudobulk analysis with DESeq2. d–f qRT-PCR of the calcineurin catalytic subunit Ppp3ca mRNA levels in d hippocampus, e cortex, and f striatum. Statistical analysis is by unpaired t-test, two-tailed (n = 4 animals; N = 3 technical replicates; ns: p > 0.05, **: p ≤ 0.01). All bar graphs represent mean ± SEM

Article Snippet: Sections were treated for endogenous peroxidases with 3% hydrogen peroxide in PBS (pH 7.4) for 30 min at room temperature, blocked in 5% non-fat milk in PBS for one hour at room temperature, and incubated with one of the following primary antibodies overnight at 4 °C: calcineurin (CN-A1 mAb, Rockland 200-301-P23, 1:500), NeuN (A60 mAb, MilliporeSigma MAB377, 1:2000) and pTDP-43 (S409/410, clone 1D3, gift from Dr. Manuela Neumann, 1:400) [ ].

Techniques: RNA Sequencing, Control, Expressing, Generated, Quantitative RT-PCR, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Calcineurin depletion coincides with phosphorylated TDP-43 deposition in a mouse model of ALS/FTLD-TDP

doi: 10.1186/s40478-025-02192-9

Figure Lengend Snippet: Activation of calcineurin protects against TDP-43 phosphorylation in mouse primary neurons. C57BL/6 mouse primary cortical neurons were cultured for 7 days and pretreated with either 0 µM or 250 µM nickel chloride (NiCl 2 ) for 3 h to activate calcineurin. Cells were then exposed to 75 µM ethacrynic acid (EA) for 3 h to induce TDP-43 phosphorylation. a Representative immunoblots for total TDP-43, phosphorylated TDP-43 (pTDP-43), and actin. b – c Densitometry analysis of b total TDP-43 and c phosphorylated TDP-43 chemiluminescence signals normalized to actin and to control samples within each set. Statistical analysis is by paired t -test, two-tailed ( n = 4 independent experiments; ns: p > 0.05 and **: p ≤ 0.01). All bar graphs represent mean ± SEM

Article Snippet: Sections were treated for endogenous peroxidases with 3% hydrogen peroxide in PBS (pH 7.4) for 30 min at room temperature, blocked in 5% non-fat milk in PBS for one hour at room temperature, and incubated with one of the following primary antibodies overnight at 4 °C: calcineurin (CN-A1 mAb, Rockland 200-301-P23, 1:500), NeuN (A60 mAb, MilliporeSigma MAB377, 1:2000) and pTDP-43 (S409/410, clone 1D3, gift from Dr. Manuela Neumann, 1:400) [ ].

Techniques: Activation Assay, Phospho-proteomics, Cell Culture, Western Blot, Control, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Calcineurin depletion coincides with phosphorylated TDP-43 deposition in a mouse model of ALS/FTLD-TDP

doi: 10.1186/s40478-025-02192-9

Figure Lengend Snippet: Activation of calcineurin protects against TDP-43 neurotoxicity in C. elegans . a A gain-of-function mutation in the C. elegans calcineurin homolog [ tax-6(jh107) ] improves TDP-43 transgenic C. elegans (TDP-43 Tg) motor dysfunction. Dispersal velocity of developmentally staged L4 larvae was measured by calculating the linear distance traveled from a designated central starting point over time. Statistical analysis is by unpaired t -test, two-tailed ( n > 100 animals; N = 3 independent experiments; ****: p ≤ 0.0001). b A transgene overexpressing wildtype TAX-6 ( tax-6 o/ex ) improves TDP-43 Tg motor dysfunction, as measured by dispersal velocity. Statistical analysis is by unpaired t -test, two-tailed ( n > 90 animals; N = 3 independent experiments, **: p ≤ 0.01). c Representative electropharyngeogram (EPG) recording traces from non-Tg, TDP-43 Tg, and TDP-43 Tg; tax-6(jh107) animals. Traces are 2 s; bar is 0.4 s. Frequency ( d ) and duration ( e ) of C. elegans pharyngeal pumping was evaluated using EPG recordings of neuron and muscle electrical signaling. tax-6(jh107) partially restores pharyngeal pumping frequency and restores pharyngeal pumping duration in TDP-43 Tg animals. Statistical analysis is by one-way ANOVA, followed by Tukey’s post-test ( n ≥ 29 animals; N = 3 independent experiments; ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ****: p ≤ 0.0001). f GFP-labeled D-type GABAergic motor neurons were counted during late day 1 of adulthood in vivo in living animals. TDP-43 Tg animals had an average of 14.6 out of 19 total neurons (13 VD + 6 DD type neurons). TDP-43 Tg; tax-6(jh107) gain-of-function animals averaged 16.1 neurons per animal. Statistical analysis is by unpaired t -test, two-tailed ( n ≥ 25 animals; ****: p ≤ 0.0001). All bar graphs represent mean ± SEM

Article Snippet: Sections were treated for endogenous peroxidases with 3% hydrogen peroxide in PBS (pH 7.4) for 30 min at room temperature, blocked in 5% non-fat milk in PBS for one hour at room temperature, and incubated with one of the following primary antibodies overnight at 4 °C: calcineurin (CN-A1 mAb, Rockland 200-301-P23, 1:500), NeuN (A60 mAb, MilliporeSigma MAB377, 1:2000) and pTDP-43 (S409/410, clone 1D3, gift from Dr. Manuela Neumann, 1:400) [ ].

Techniques: Activation Assay, Mutagenesis, Transgenic Assay, Two Tailed Test, Labeling, In Vivo

Journal: Cell Death & Disease

Article Title: miR-146a facilitates osteoarthritis by regulating cartilage homeostasis via targeting Camk2d and Ppp3r2

doi: 10.1038/cddis.2017.146

Figure Lengend Snippet: MiR-146a targets Tgif1, Camk2d and Ppp3r2. ( a ) Total RNA of mouse articular chondrocytes transfected with miR-146a inhibitor or control (NC) inhibitor was used for microarray analysis. Twelve genes were validated based on the seed region of miR-146a in 3′-UTR of candidate gene that is evolutionarily conserved in mammals. The gradient colors and numbers indicate fold change of gene expression in mouse chondrocytes transfected with the miR-146a inhibitor compared with control, as determined by microarray. Fold 146a/NC , fold change of gene expression in mouse chondrocytes transfected with miR-146a inhibitor compared with control inhibitor. ( b ) qPCR analysis of mRNA expression of candidate target genes in mouse articular chondrocytes transfected with miR-146a mimic or control mimic plasmid ( n =3 for each treatment). ( c and d ) Luciferase activity analysis in HEK293T cells that were co-transfected with the indicated 3′-UTR reporter ( c ) or with the indicated WT or mutant 3′-UTR reporter ( d ) along with miR-146a mimic or control mimic plasmid ( n =3 for each treatment). The results were normalized by internal firefly luciferase readout. ( e-g ) qPCR analysis of mRNA expression of Tgif1 ( e ), Camk2d ( f ) and Ppp3r2 ( g ) in articular cartilage of mice (WT, n =6; miR-146a KO, n =8) collected at 4 weeks after DMM surgery. ( h and i ) Western blot analysis of endogenous Sox9 and Col2a1 protein expression in mouse chondrocytes that were transfected with indicated expression vector constructs ( h ) or with gene-specific siRNAs as well as a scrambled control siRNA (siRNA-NC). For quantification, protein expression was normalized by the protein amount in the first lane. ( i ) Three independent experiments were performed with similar results in b-d . Relative mRNA expression was normalized to the expression of GAPDH. Data are mean±S.D. * P <0.05, ** P <0.01 versus control group (Student's t -test) in b , c and d and Mann–Whitney test in e-g . NS, not significant between the indicated groups

Article Snippet: Sections were blocked with 5% goat serum, 0.1% tween-20 in phosphate-buffered saline (PBS) for 30 min at room temperature, and then incubated with following primary antibodies to Mmp13 (1:200, ab39012; Abcam, Cambridge, UK), type X collagen (1:1000, ab58632, Abcam), GFP (1:1000, ab290, Abcam), Camk2d (1:100, 20667-1-AP; ProteinTech Group, Chicago, IL, USA), Ppp3r2 (1:100, 14005-1-AP, ProteinTech Group), Nfatc1 (1:50, ab175134, Abcam), Nfatc2 (1:100, 22023-1-AP, ProteinTech Group) and isotype control antibody (ab27478, Abcam) overnight at 4 °C.

Techniques: Transfection, Control, Microarray, Gene Expression, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Mutagenesis, Western Blot, Construct, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: miR-146a facilitates osteoarthritis by regulating cartilage homeostasis via targeting Camk2d and Ppp3r2

doi: 10.1038/cddis.2017.146

Figure Lengend Snippet: Camk2d and Ppp3r2 are direct targets of miR-146a in vivo . ( a-e ) Immunohistochemical staining and quantitative analysis of the percentage of Camk2d, Ppp3r2, Nfatc1 and Nfatc2-positive chondrocytes in knee joint cartilage of 3- and 12-month-old WT or miR-146a KO mice. n =8 per group. Scale bars, 100 μ m. ( f-h ) Immunohistochemical staining and quantitative analysis of the percentage of Camk2d and Ppp3r2-positive chondrocytes in knee joint cartilage harvested from the indicated groups. n =8–9 per group. Scale bars, 100 μ m. Data are the mean±S.D. * P <0.05, ** P <0.01 (Mann–Whitney test). NS, not significant between the indicated groups

Article Snippet: Sections were blocked with 5% goat serum, 0.1% tween-20 in phosphate-buffered saline (PBS) for 30 min at room temperature, and then incubated with following primary antibodies to Mmp13 (1:200, ab39012; Abcam, Cambridge, UK), type X collagen (1:1000, ab58632, Abcam), GFP (1:1000, ab290, Abcam), Camk2d (1:100, 20667-1-AP; ProteinTech Group, Chicago, IL, USA), Ppp3r2 (1:100, 14005-1-AP, ProteinTech Group), Nfatc1 (1:50, ab175134, Abcam), Nfatc2 (1:100, 22023-1-AP, ProteinTech Group) and isotype control antibody (ab27478, Abcam) overnight at 4 °C.

Techniques: In Vivo, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: miR-146a facilitates osteoarthritis by regulating cartilage homeostasis via targeting Camk2d and Ppp3r2

doi: 10.1038/cddis.2017.146

Figure Lengend Snippet: Schematic model of the role of miR-146a in the pathogenesis of OA. MiR-146a is induced in chondrocytes in an NF-кB-dependent manner by pro-inflammatory cytokines stimulation. Once activated, miR-146a disrupts cartilage homeostasis via targeting Camk2d and Ppp3r2, which are associated with cartilage anabolism, thereby inhibiting the activation of downstream NFAT pathway

Article Snippet: Sections were blocked with 5% goat serum, 0.1% tween-20 in phosphate-buffered saline (PBS) for 30 min at room temperature, and then incubated with following primary antibodies to Mmp13 (1:200, ab39012; Abcam, Cambridge, UK), type X collagen (1:1000, ab58632, Abcam), GFP (1:1000, ab290, Abcam), Camk2d (1:100, 20667-1-AP; ProteinTech Group, Chicago, IL, USA), Ppp3r2 (1:100, 14005-1-AP, ProteinTech Group), Nfatc1 (1:50, ab175134, Abcam), Nfatc2 (1:100, 22023-1-AP, ProteinTech Group) and isotype control antibody (ab27478, Abcam) overnight at 4 °C.

Techniques: Activation Assay