rabbit polyclonal sirt2 antibody  (Millipore)

Journal: iScience

Article Title: Mitochondrial glycerol 3-phosphate dehydrogenase deficiency exacerbates lipotoxic cardiomyopathy

doi: 10.1016/j.isci.2024.109796

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-SIRT2 antibody , Proteintech , Cat# 19655-1-AP; RRID: AB_2878592.

Techniques: Virus, Recombinant, Modification, Transfection, Labeling, XF Assay, Isolation, Staining, Bicinchoninic Acid Protein Assay, Immunoprecipitation, Plasmid Preparation, Control, Software

Journal: eLife

Article Title: SIRT2 inhibition protects against cardiac hypertrophy and ischemic injury

doi: 10.7554/eLife.85571

Figure Lengend Snippet: ( A ) SIRT1, SIRT2, SIRT3, and SIRT6 in mouse hearts after trans-aortic constriction (TAC). ( B ) SIRT2 in human hearts from healthy patients and patients with dilated cardiomyopathy. ( C ) SIRT2 protein levels in the hearts of control individual and patients with ischemic heart failure. *p<0.05 by Student’s t-test. Data presented as mean ± SEM. Figure 1—source data 1. SIRT1, -2, -3, and -6 after sham and trans-aortic constriction (TAC) surgery as shown in . Figure 1—source data 2. SIRT2 in non-failing and failing human hearts as shown in . Figure 1—source data 3. SIRT2 in non-failing and ischemic human hearts as shown in . Figure 1—source data 4. Full gels for . Figure 1—source data 5. Full gels for unedited. Figure 1—source data 6. Full gels for unedited.

Article Snippet: Antibody , Rabbit polyclonal SIRT2 antibody , Sigma , S8447 , WB (1:1000).

Techniques:

Journal: eLife

Article Title: SIRT2 inhibition protects against cardiac hypertrophy and ischemic injury

doi: 10.7554/eLife.85571

Figure Lengend Snippet: Sirt2 -/- and wild-type (WT) littermates were subjected to TAC and ejection fraction (EF) ( A ), fractional shortening (FS) ( B ), and interventricular septal thickness during diastole ( C ) were assessed 4 weeks later (N=6–9). ( D–F ) Representative hearts ( D ), HW/BW ( E ) (N=3–5), H&E staining, ( F ) and the summary of cross-sectional area of cardiomyocytes ( G ) in WT and Sirt2 -/- hearts (N=20 cardiomyocytes), *p<0.05 by one-way ANOVA and post hoc Tukey analysis ( A, B, C, and E ) and unpaired Student’s t-test ( G ). Bars represent group mean. Figure 2—source data 1. Ejection fraction (EF) in wild-type (WT) and Sirt2 -/- mice after sham or trans-aortic constriction (TAC) as shown in . Figure 2—source data 2. Fractional shortening (FS) in wild-type (WT) and Sirt2 -/- mice after sham or trans-aortic constriction (TAC) as shown in . Figure 2—source data 3. Interventricular septal (IVS) thickness diastole in wild-type (WT) and Sirt2 -/- mice after sham or trans-aortic constriction (TAC) as shown in . Figure 2—source data 4. HW/BW in wild-type (WT) and Sirt2 -/- mice after sham or trans-aortic constriction (TAC) as shown in . Figure 2—source data 5. CSA in wild-type (WT) and Sirt2 -/- hearts as shown in .

Article Snippet: Antibody , Rabbit polyclonal SIRT2 antibody , Sigma , S8447 , WB (1:1000).

Techniques: Staining

Journal: eLife

Article Title: SIRT2 inhibition protects against cardiac hypertrophy and ischemic injury

doi: 10.7554/eLife.85571

Figure Lengend Snippet: Ejection fraction (EF) and fractional shortening (FS) in Sirt2 f/f and cs- Sirt2 -/- mice 7 ( A ) and 14 days ( B ) after TAC (N=5–9). ( C,D ) mRNA levels of Anf ( C ) and Bnp ( D ) in the hearts of Sirt2 f/f and cs- Sirt2 -/- mice 4 weeks after TAC (N=7–8). ( E ) EF and FS in Sirt2 f/f and cs- Sirt2 -/- mice 7 and 14 days after I/R (N=4). ( F ) Necrotic area (representing the degree of ischemic damage) in Sirt2 f/f and cs- Sirt2 -/- mice 14 days after MI. *p<0.05 by ANOVA for panels A and B, and Student’s t-test was used for panels C and D. Data are presented as mean ± SEM. Figure 4—source data 1. Ejection fraction (EF) and fractional shortening (FS) in Sirt2 f/f and cs- Sirt2 -/- mice 7 days after ischemia-reperfusion (I/R) as shown in . Figure 4—source data 2. Ejection fraction (EF) and fractional shortening (FS) in Sirt2 f/f and cs- Sirt2 -/- mice 14 days after ischemia-reperfusion (I/R) as shown in . Figure 4—source data 3. Nppa mRNA in Sirt2 f/f and cs- Sirt2 -/- hearts as shown in . Figure 4—source data 4. Nppb mRNA in Sirt2 f/f and cs- Sirt2 -/- hearts as shown in . Figure 4—source data 5. Echo parameters in Sirt2 f/f and cs- Sirt2 -/- hearts as shown in .

Article Snippet: Antibody , Rabbit polyclonal SIRT2 antibody , Sigma , S8447 , WB (1:1000).

Techniques:

Journal: eLife

Article Title: SIRT2 inhibition protects against cardiac hypertrophy and ischemic injury

doi: 10.7554/eLife.85571

Figure Lengend Snippet: ( A ) Co-immunoprecipitation (IP) of SIRT2 and NRF2 in extracts of hearts from wild-type (WT) mice. ( B ) Endogenous NRF2 acetylation levels in the hearts of WT and Sirt2 -/- mice at the baseline. Acetylated proteins were IPed by anti-acetyl antibody followed by immunoblotting with anti-NRF2 antibody. ( C ) NRF2 protein levels in neonatal rat cardiomyocytes (NRCMs) treated with Sirt2 siRNA. ( D ) NRF2 protein levels in H9c2 cells treated with control or Sirt2 siRNA and harvested at different time points after treatment with 100 µg/ml of CHX. ( E ) NRF2 protein levels in the nucleus in NRCMs treated with control or Sirt2 siRNA. ( F–H ) mRNA levels of NRF2 target genes in pentose phosphate pathway ( F ), quinone and glutathione-based detoxification ( G ), thioredoxin production ( H ) in H9c2 cells overexpressing empty vector (white bars) or SIRT2 (gray bars). *p<0.05 by Student’s t-test. Figure 5—source data 1. mRNA with overexpression of EV or SIRT2 as shown in . Figure 5—source data 2. mRNA with overexpression of EV or SIRT2 as shown in . Figure 5—source data 3. mRNA with overexpression of EV or SIRT2 as shown in . Figure 5—source data 4. Uncropped gels for . Figure 5—source data 5. Uncropped gels for unedited. Figure 5—source data 6. Uncropped gels for unedited.

Article Snippet: Antibody , Rabbit polyclonal SIRT2 antibody , Sigma , S8447 , WB (1:1000).

Techniques: Immunoprecipitation, Western Blot, Plasmid Preparation, Over Expression

Journal: eLife

Article Title: SIRT2 inhibition protects against cardiac hypertrophy and ischemic injury

doi: 10.7554/eLife.85571

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal SIRT2 antibody , Sigma , S8447 , WB (1:1000).

Techniques: Knock-Out, Activity Assay, In Vivo, Protease Inhibitor, Transfection, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Extraction, Staining, Sequencing, Recombinant, Plasmid Preparation, Software

Journal: Journal of Neuroimmune Pharmacology

Article Title: SIRT2 Inhibition Rescues Neurodegenerative Pathology but Increases Systemic Inflammation in a Transgenic Mouse Model of Alzheimer’s Disease

doi: 10.1007/s11481-023-10084-9

Figure Lengend Snippet: SIRT2 inhibition increases microglial phagocytosis of methoxy-labeled Aβ. ( a ) Experimental design for quantitative in vivo assessment of amyloid-beta phagocytic capacity and gating strategy to identify CD11b + CD45 low microglia. SSC: side scatter; FSC: forward scatter. ( b ) Quantification of Aβ phagocytosis by flow cytometry of microglia isolated from vehicle or 33i treated 8 months-old APP/PS1 mice 3 h after intraperitoneal injection of methoxy-X04 (*p < 0.05, Student’s t-test). Results are shown as mean ± SEM (n = 5–6 animals per group). ( c ) Representative FACS plots demonstrating the engulfment of Aβ by microglia isolated from APP/PS1 mice upon treatment with vehicle or 33i. Wild-type mice (WT) injected with methoxy-X04 were used to determine the methoxy-X04-threshold for non-phagocytic cells

Article Snippet: The trans-blots were blocked in TBS-Tween containing 5% powder milk for 1 h. Membranes were probed overnight at 4 °C with rabbit polyclonal antibody anti-SIRT2 (1:1000; cat# S8447, Sigma-Aldrich).

Techniques: Inhibition, Labeling, In Vivo, Flow Cytometry, Isolation, Injection

Journal: Journal of Neuroimmune Pharmacology

Article Title: SIRT2 Inhibition Rescues Neurodegenerative Pathology but Increases Systemic Inflammation in a Transgenic Mouse Model of Alzheimer’s Disease

doi: 10.1007/s11481-023-10084-9

Figure Lengend Snippet: SIRT2 inhibition induces peripheral inflammation. ( a ) Weekly body weight monitoring of WT and APP/PS1 mice during the treatment. Glucose ( b ) and Insulin ( c ) tolerance tests. 33i treatment for two months in WT and APP/PS1 mice did not have any significant effect on glucose and insulin tolerance (n = 12–14 animals per group). ( d ) Gene expression of Il-1β (F = 7.529, *p < 0.05, main effect of treatment; F = 5.532, #p < 0.05, main effect of genotype, two-way ANOVA, n = 5–6 mice per group) and ( e ) protein expression of IL-1β (F = 50.13, ***p < 0.01, main effect of treatment; F = 4.978, #p < 0.05, main effect of genotype, two-way ANOVA, n = 7–8 animals per group) in white adipose tissue of WT and APP/PS1 mice. Note that 33i treatment increased levels of this pro-inflammatory cytokine in WT and APP/PS1 animals. Peripheral gene expression of ( f ) Tnf-α (F = 5.201, *p < 0.05, main effect of treatment; F = 21.11, ###p < 0.001, main effect of genotype, two-way ANOVA) and ( g ) Tgf-β (F = 11.46, ##p < 0.01, main effect of genotype, two-way ANOVA) (n = 5–6 animals per group). 36b4 was used as an internal control. Serum levels of the cytokines ( h ) IL-6 (F = 18.76, ***p < 0.001, main effect of treatment, two-way ANOVA), ( i ) MCP-1 (F = 7.782, *p < 0.05, main effect of treatment, two-way ANOVA) and ( j ) TNF (F = 8.901, **p < 0.01, main effect of treatment, two-way ANOVA) (n = 5–8 mice per group). Results are shown as mean ± SEM

Article Snippet: The trans-blots were blocked in TBS-Tween containing 5% powder milk for 1 h. Membranes were probed overnight at 4 °C with rabbit polyclonal antibody anti-SIRT2 (1:1000; cat# S8447, Sigma-Aldrich).

Techniques: Inhibition, Expressing

Journal: Journal of Neuroimmune Pharmacology

Article Title: SIRT2 Inhibition Rescues Neurodegenerative Pathology but Increases Systemic Inflammation in a Transgenic Mouse Model of Alzheimer’s Disease

doi: 10.1007/s11481-023-10084-9

Figure Lengend Snippet: Peripheral SIRT2 inhibition impairs memory and increases systemic inflammation. ( a ) Habituation phase of the MWM. ( b ) Escape latency in the acquisition phase of the MWM and corresponding area under the curve (AUC) of the acquisition curve (F = 6.716, *p < 0.05 main effect of treatment; F = 6.580, #p < 0.05 main effect of genotype, two-way ANOVA, n = 6–8 animals per group). Note that AGK-2 treatment worsened learning capacities in both WT and APP/PS1 mice. ( c ) Representation of the percentage of time spent in the correct quadrant in the retention phase of the MWM (5 th Day: F = 4.474 *p < 0.05, main effect of treatment; 8 th Day: F = 4.854, #p < 0.05, main effect of genotype, two-way ANOVA). ( d ) Representative hippocampal sections of β-amyloid plaques stained with 6E10 antibody in brain slices (left) and amyloid burden quantification (right) in 8 months-old APP/PS1 mice treated for two months with vehicle or AGK-2 (n = 3 animals per group, 2 sections including hippocampus and frontal cortex per animal) Scale bar = 500 µm. Glucose ( e ) and Insulin ( f ) tolerance tests. No significant differences were observed between vehicle or AGK-2 treated animals (n = 5–9 mice per group). Peripheral protein expression of ( g ) IL-1β (F = 5.951, *p < 0.05, main effect of treatment, two-way ANOVA) and gene expression of ( h ) Il-1β (F = 16.33, ***p < 0.001, main effect of treatment, two-way ANOVA), ( i ) Tnf-α (F = 19.60, ***p < 0.001, main effect of treatment, two-way ANOVA) and ( j ) Tgf-β (F = 11.49, **p < 0.01, main effect of treatment, two-way ANOVA) (n = 6 animals per group). 36b4 was used as an internal control. Serum levels of the cytokines ( k ) IL-6 (F = 10.80, ***p < 0.001, main effect of treatment, two-way ANOVA), ( l ) MCP-1 and ( m ) TNF (F = 5.926, *p < 0.05, main effect of treatment, two-way ANOVA) (n = 5–8 mice per group). Results are shown as mean ± SEM

Article Snippet: The trans-blots were blocked in TBS-Tween containing 5% powder milk for 1 h. Membranes were probed overnight at 4 °C with rabbit polyclonal antibody anti-SIRT2 (1:1000; cat# S8447, Sigma-Aldrich).

Techniques: Inhibition, Staining, Expressing

Journal: Journal of Neuroimmune Pharmacology

Article Title: SIRT2 Inhibition Rescues Neurodegenerative Pathology but Increases Systemic Inflammation in a Transgenic Mouse Model of Alzheimer’s Disease

doi: 10.1007/s11481-023-10084-9

Figure Lengend Snippet: SIRT2 is increased in postmortem brain tissue from Alzheimer’s disease patients but not in serum. ( a ) Gene expression of SIRT2 in frontal cortex of postmortem control and Alzheimer’s disease (AD) human samples (*p < 0.05, Student’s t-test). β-ACTIN was used as internal control (n = 10 samples per group). ( b ) Representative western blot images (top) and SIRT2 protein levels quantification (bottom) in frontal cortex of postmortem control and AD human samples (*p < 0.05, Student’s t-test). β-ACTIN was used as internal control (n = 7 samples per group). ( c ) No significant differences between both groups were found when SIRT2 was analysed in serum samples (n = 24 samples per group)

Article Snippet: The trans-blots were blocked in TBS-Tween containing 5% powder milk for 1 h. Membranes were probed overnight at 4 °C with rabbit polyclonal antibody anti-SIRT2 (1:1000; cat# S8447, Sigma-Aldrich).

Techniques: Expressing, Western Blot

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a Western blotting was performed to examine the protein expression of SIRT2 in wild-type (WT) and SIRT2 knockout (KO) mice. b Reverse-transcription PCR (RT-PCR) was used to detect the mRNA expression of Sirt2 in WT and SIRT2 KO mice. c Immunostaining was performed to detect tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc) in MPTP-treated WT and SIRT2 KO mice (the control group was treated with normal saline (NS)). The scale bar represents 200 μm. d The number of TH-positive neurons in the SNpc was counted ( n = 3). e The open field test and f Rotarod test were used to examine MPTP-treated WT and SIRT2 KO mice ( n = 15). g RT-PCR was employed to assess the mRNA expression of α-synuclein and Sirt2 in WT mice, α-synuclein-A30P*A53T transgenic (TG) mice, and α-synuclein-A30P*A53T transgenic mice with SIRT2 knockout (TG-KO). h Immunostaining was performed to detect TH-positive neurons in the SNpc in WT, TG, and TG-KO mice. The scale bar represents 200 μm. i Numbers of TH-neurons in the SNpc were counted stereologically ( n = 3). j The open field test and k rotarod test were used to detect motor deficits in WT, TG, and TG-KO mice ( n = 8). All data are presented as the means ± SD. Statistical analyses were conducted using two-way ANOVA followed by Tukey’s post hoc test in ( d – f ). Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test in ( i – k ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: Western Blot, Expressing, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Transgenic Assay

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a The mRNA levels of SIRT2 in the SNpc were examined using RT-PCR in mice treated with normal saline (NS) or MPTP. b Quantification of SIRT2 mRNA levels shown in ( a ) ( n = 3). c The protein levels of SIRT2 in the SNpc were examined using western blotting in mice treated with NS or MPTP. d Quantification of SIRT2 protein levels shown in ( c ) ( n = 3). e – h mRNA and protein levels of SIRT2 in the SNpc assessed were assessed using RT-PCR and western blotting ( n = 3), respectively, in WT and α-synuclein-A30P*A53T transgenic (TG) mice. i – l mRNA and protein levels of SIRT2 were examined in primary culture neurons treated with 50 μM MPP + for 0, 6, 12, and 24 h using RT-PCR and western blotting ( n = 3). m – p SIRT2 expression was examined in SY5Y cells stably expressing GFP vector, GFP-α-synuclein, GFP-α-synuclein-A53T, and GFP-α-synuclein-A30P*A53T ( n = 3). All data are presented as the means ± SD. Statistical analyses were conducted using an unpaired t -test in ( b ) and ( d ). Statistical analyses were conducted using two-way ANOVA followed by Tukey’s post hoc test in ( f ) and ( h ). Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test in ( j , l , n , p ). None of the differences were significant.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transgenic Assay, Expressing, Stable Transfection, Plasmid Preparation

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a , b Subcellular localization of SIRT2 was monitored using dual immunolabeling for TH (red) and SIRT2 (green) in the SNpc in mice treated with normal saline (NS) or MPTP ( a ) as well as in WT and α-synuclein-A30P*A53T transgenic mice (TG) ( b ). c Immunofluorescence assays for SIRT2 (green) were conducted in primary culture neurons treated with 50 μM MPP + for 24 h. d The localization of SIRT2 was detected using nuclear/cytosolic immunoblotting in primary culture neurons treated with 50 μM MPP + for 0, 6, 12, and 24 h. e Quantification of SIRT2 protein levels in the cytoplasm and nucleus (separate from d ). f SH-SY5Y cells were transiently transfected with GFP vector (green) or GFP-α-synuclein-A30P*A53T (green) plasmids for 24 h and subsequently immunostained for SIRT2 (red) and observed using confocal microscopy. g Subcellular localization of SIRT2 examined using dual immunolabeling for TH (red) and SIRT2 (green) in the mouse SNpc 35 days after the striatal stereotactic injection of 2 μl (per side) normal saline (NS), or α-synuclein PFF (Abcam, ab246002, 1 μg/μl). h Localization of SIRT2 detected using nuclear/cytoplasmic immunofluorescence staining in primary culture neurons treated with 4 μg/ml PFF for 7 days. i Localization of SIRT2 detected using nuclear/cytosolic immunoblotting in primary culture neurons treated with 4 μg/ml PFF for 7 days. j Quantification of SIRT2 protein levels in the cytoplasm and nucleus (separate from [ i ]). The scale bar represents 20 μm. All data are presented as the means ± SD. Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test in ( e ). Statistical analyses were conducted using two-way ANOVA followed by Tukey’s post hoc test in ( j ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: Immunolabeling, Transgenic Assay, Immunofluorescence, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy, Injection, Staining

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a Primary culture neurons were transiently transfected with GFP vector or GFP-NLS-SIRT2 plasmids and subsequently stained using anti-SIRT2 antibodies (green), propidium iodide (PI) (red), and DAPI (blue). The scale bar represents 10 μm. b GFP-positive cells (green) that also showed positive PI staining (red) were counted as dead neurons. The percentage of PI-positive cells (red) among at least 100 GFP-positive cells was measured for each group ( n = 3). All data are presented as the means ± SD. Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test, and ** represents P < 0.01. c HEK293 cells were transfected with GFP vector and GFP-NLS-SIRT2 plasmids for 24 h and subsequently collected for RNA-seq. The differentially expressed genes between the two groups are illustrated using a heatmap ( n = 3). The colors indicate relative expression levels (red, high expression; green, low expression). d Gene Ontology (GO) terms. Frequency of sequences with assigned GO terms for the biological process category across the different samples. e KEGG pathway enrichment. Biological pathways associated with the differentially expressed genes in the different samples.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: Transfection, Plasmid Preparation, Staining, RNA Sequencing Assay, Expressing

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a Co-Immunoprecipitation (Co-IP) was performed to identify whether endogenous SIRT2 interacts with Cdk5. b A GST-pull-down assay was used to verify whether exogenous SIRT2 can bind to Cdk5. c Cultured primary neurons were pretreated with 10 μM roscovitine (ROS) for 0.5 h and subsequently treated with 50 μM MPP + for 24 h. The cytoplasmic and nuclear distribution of SIRT2 was detected using immunoblotting. d Quantification of SIRT2 protein levels in the cytoplasm and nucleus shown in c ( n = 3). e Immunofluorescence staining for SIRT2 (green) was performed to observe the distribution of SIRT2 in primary culture neurons subjected to ROS stress. The scale bar represents 10 μm. All data are presented as the means ± SD. Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test in d . * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Pull Down Assay, Cell Culture, Western Blot, Immunofluorescence, Staining

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a Liquid chromatography-mass spectrometry after a kinase assay in vitro demonstrated that the Ser331 and b Ser335 sites of SIRT2 were phosphorylated by Cdk5. c Phosphorylation levels of SIRT2 were detected in forebrain cortical tissue obtained from forebrain neuron-specific Cdk5 knockout mice and WT mice. Phosphorylation of SIRT2 was detected in primary culture neurons using immunoprecipation and western blotting with anti-phosS/TP and anti-SIRT2 antibodies. d SDS-PAGE used for the Cdk5/p35 kinase assay in vitro. Purified GST-SIRT2 WT, S331A, S335A, and S331AS335A (AA) fusion proteins were mixed with active Cdk5/p35 and ATP, and the reaction mixture was analyzed using western blotting with anti-phosS/TP and anti-GST antibodies. e HEK293 cells were transiently transfected with Flag-SIRT2 WT or Flag-SIRT2 mutation DD (double mutations at S331D and S335D) plasmids for 24 h, and subsequently, the cytoplasmic and nuclear distribution of SIRT2 (green) was detected using immunofluorescence staining. The scale bar represents 10 μm. f Immunoblotting assays for SIRT2 was performed using an anti-Flag antibody in HEK293 cells transfected with Flag-SIRT2 WT or Flag-SIRT2-DD. g Quantification of SIRT2 protein levels shown in f ( n = 3). All data are presented as the means ± SD. Statistical analyses were conducted using two-way ANOVA followed by Tukey’s post hoc test in g . * P < 0.05, *** P < 0.001.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: Liquid Chromatography, Mass Spectrometry, Kinase Assay, In Vitro, Knock-Out, Western Blot, SDS Page, Purification, Transfection, Mutagenesis, Immunofluorescence, Staining

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a Primary culture neurons were pretreated with the SIRT2 328–339 or Scramble peptide conjugated with Myristic acid (Myr) and subsequently treated with 100 μM MPP + for 48 h. The survival percentage of neurons was detected using an MTT assay. b , c Immunostaining for TH in the SNpc, d the open field test, and e the rotarod test were performed in MPTP-treated mice who received Myr-SIRT2 328–339 at a dose of 2 mg/kg (i.p.) once per day. The scramble peptide was used as the control. The number of TH-positive neurons in the SNpc was counted (Myr-SIRT2 328–339 , n = 3; Scramble, n = 4). The behavioral tests were conducted in 10 (Myr-SIRT2 328–339 ) and 12 mice (Scramble) each. All data are presented as the means ± SD. Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test in ( a , c – e ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: MTT Assay, Immunostaining