Journal: Neurochemical Research

Article Title: Idebenone Antagonizes P53-Mediated Neuronal Oxidative Stress Injury by Regulating CD38-SIRT3 Protein Level

doi: 10.1007/s11064-024-04189-7

Figure Lengend Snippet: Prediction of potential targets of Idebenone by network pharmacology. ( A )Cytoscape to visualize potential targets of Idebenone. ( B ) Potential targets of Idebenone and PPI networks of SIRT3

Article Snippet: Rat anti-mouse P53 protein polyclonal antibody (Bioss Biotech Co., LTD, China), rabbit anti-mouse Caspase3 polyclonal antibody (Bioss Biotech Co., LTD, China), rabbit anti-mouse SIRT3 polyclonal antibody (Bioss Biotech Co., LTD, China), rabbit anti-mouse P53-AC monoclonal antibody (Abcam, England), and rabbit anti-mouse β-actin monoclonal antibody (Bioss Biotech Co., LTD, China) were separately incubated with protein-transferred membranes overnight at 4℃, and then they were washed with TBST for 3 times.

Techniques:

Journal: Neurochemical Research

Article Title: Idebenone Antagonizes P53-Mediated Neuronal Oxidative Stress Injury by Regulating CD38-SIRT3 Protein Level

doi: 10.1007/s11064-024-04189-7

Figure Lengend Snippet: Idebenone Upregulates SIRT3 Expression in H 2 O 2 -damaged HT22 Cells. ( A ) The expression of SIRT3 protein was detected by western blot. ( B ) The expression of SIRT3 protein was detected by real-time PCR. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: Rat anti-mouse P53 protein polyclonal antibody (Bioss Biotech Co., LTD, China), rabbit anti-mouse Caspase3 polyclonal antibody (Bioss Biotech Co., LTD, China), rabbit anti-mouse SIRT3 polyclonal antibody (Bioss Biotech Co., LTD, China), rabbit anti-mouse P53-AC monoclonal antibody (Abcam, England), and rabbit anti-mouse β-actin monoclonal antibody (Bioss Biotech Co., LTD, China) were separately incubated with protein-transferred membranes overnight at 4℃, and then they were washed with TBST for 3 times.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Neurochemical Research

Article Title: Idebenone Antagonizes P53-Mediated Neuronal Oxidative Stress Injury by Regulating CD38-SIRT3 Protein Level

doi: 10.1007/s11064-024-04189-7

Figure Lengend Snippet: Idebenone Induces the Expression of SIRT3 in SIRT3-Down-Regulated HT22 Cells and the Deacetylation of P53Ac. ( A ) The expression of P53Ac protein was detected by western blot after SIRT3 down-regulated in various groups. * p < 0.05, ** p < 0.01 and *** p < 0.001 ( B ) The expression of SIRT3 protein was detected by western blot after SIRT3 down-regulated in various groups. * p < 0.05, ** p < 0.01 and *** p < 0.001 ( C ) The expression of SIRT3 was detected by real-time PCR after SIRT3 down-regulated. * p < 0.05, ** p < 0.01 and *** p < 0.001 ( D ) The expression of P53 protein was detected by real-time PCR after SIRT3 down-regulated in various groups. * p < 0.05, ** p < 0.01 and *** p < 0.001 ( E ) The expression of SIRT3 protein was detected by real-time PCR after SIRT3 down-regulated in various groups. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: Rat anti-mouse P53 protein polyclonal antibody (Bioss Biotech Co., LTD, China), rabbit anti-mouse Caspase3 polyclonal antibody (Bioss Biotech Co., LTD, China), rabbit anti-mouse SIRT3 polyclonal antibody (Bioss Biotech Co., LTD, China), rabbit anti-mouse P53-AC monoclonal antibody (Abcam, England), and rabbit anti-mouse β-actin monoclonal antibody (Bioss Biotech Co., LTD, China) were separately incubated with protein-transferred membranes overnight at 4℃, and then they were washed with TBST for 3 times.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: CNS Neuroscience & Therapeutics

Article Title: Citrate synthase lysine K215 hypoacetylation contributes to microglial citrate accumulation and pro‐inflammatory functions after traumatic brain injury

doi: 10.1111/cns.14567

Figure Lengend Snippet: Sirt3 is responsible for Cs deacetylation in primary microglia during neuroinflammation. (A, E) Protein interaction between Cs and Sirt3, and Sirt3 expression were analyzed by immunoprecipitation and western blotting in primary microglia expressing wildtype Cs after LPS stimulation ( n = 3). Two‐tailed unpaired t ‐test was used. (B, F) Protein interaction between Cs and Sirt3, and Sirt3 expression were analyzed by immunoprecipitation and western blotting in NIH/3 T3 cells overexpressing Sirt3‐3xFlag ( n = 3). (C) Protein interaction between Cs and Sirt3 was analyzed by immunoprecipitation and western blotting in primary microglia overexpressing Sirt3‐3xFlag ( n = 3). Two‐tailed unpaired t ‐test was used. (D, G) Cs acetylation in primary microglia with wildtype Sirt3, Sirt3 knockdown, or Sirt3‐3xFlag overexpression after LPS stimulation was analyzed by immunoprecipitation and western blotting ( n = 3). One‐way ANOVA and Tukey's multiple comparisons test were used. Bar graphs are represented as mean ± SD. **** p < 0.0001, *** p < 0.001, ** p < 0.005, * p < 0.05; KD, knockdown; ns, no significance; OE: overexpression.

Article Snippet: The membrane was blocked with 5% skim milk for 2 h at room temperature and incubated with the following primary antibodies for 12 h at 4°C: rabbit anti‐Cs antibody (1:1000; Abcam, ab96600); rabbit anti‐Sirt3 (1:1000; Abcam, ab189860); HRP‐conjugated rabbit anti‐β‐tubulin (1:10,000; Abcam, ab21058).

Techniques: Expressing, Immunoprecipitation, Western Blot, Two Tailed Test, Over Expression

Journal: CNS Neuroscience & Therapeutics

Article Title: Citrate synthase lysine K215 hypoacetylation contributes to microglial citrate accumulation and pro‐inflammatory functions after traumatic brain injury

doi: 10.1111/cns.14567

Figure Lengend Snippet: Cs K215 hypoacetylation could regulate microglial pro‐inflammatory functions independent of Sirt3 expression. (A) Representative immunofluorescence and bright field images indicate primary microglial phagocytosis expressing Cs WT, Cs K215Q mutant or Sirt3 knockdown, after LPS and hesperidin administration or not ( n = 6). Arrows indicate pHRodo Zymosan Bioparticles engulfed by primary microglia. Scale bar: 20 μm. (B) The percentage of pHRodo Zymosan+ microglia in total microglia expressing Cs WT, Cs K215Q mutant, or Sirt3 knockdown, after LPS and hesperidin administration or not ( n = 6). One‐way ANOVA and Tukey's multiple comparisons test were used. (C) The mean gray value according to the red channel in was calculated by ImageJ ( n = 6). One‐way ANOVA and Tukey's multiple comparisons test were used. (D–F) IL‐1β, TNF‐α, and IL‐6 mRNA qRT‐PCR is used to evaluate microglial pro‐inflammatory cytokines ( n = 6). One‐way ANOVA and Tukey's multiple comparisons test were used. Bar graphs are represented as mean ± SD. **** p < 0.0001, *** p < 0.001, ** p < 0.005; hesp, hesperidin; KD, knockdown; ns, no significance; veh, vehicle.

Article Snippet: The membrane was blocked with 5% skim milk for 2 h at room temperature and incubated with the following primary antibodies for 12 h at 4°C: rabbit anti‐Cs antibody (1:1000; Abcam, ab96600); rabbit anti‐Sirt3 (1:1000; Abcam, ab189860); HRP‐conjugated rabbit anti‐β‐tubulin (1:10,000; Abcam, ab21058).

Techniques: Expressing, Immunofluorescence, Mutagenesis, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A) Quantitative real-time RT-PCR analysis of sirtuin gene expression after H2O2 treatment (20 μM, 6 hours) in preimplantation embryos. Sirt3 mRNA levels were significantly increased in 4-cell embryos after exposure to H2O2. Data are derived from 3 independent experiments. Statistical assessments were performed by applying Mann-Whitney U test. *P < 0.05. (B and C) Western blotting analysis showing Sirt3 protein upregulation in 4-cell embryos (B) and NIH 3T3 cells (C) after H2O2 treatment (B, 20 and 100 μM, 18 hours; C, 20 μM, 24 hours) detected by Western blot. Blotting for α-tubulin served as an internal control.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, MANN-WHITNEY, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: Intracellular injection of mRNA coding Sirt3-EGFP (A–C), Sirt1-EGFP (D–F), Sirt2-EGFP (G–I), or EGFP alone (J) was performed at the pronuclear stage, and the EGFP signals (A, D, G, and J; green) were observed at the 2-cell stage. (B, E, and H) Mitochondria were stained by MitoTracker (red) just 15 minutes before observation. (C, F, and I) Colocalization of Sirt3-EGFP signals to MitoTracker staining confirmed mitochondrial localization of Sirt3. Scale bars: 20 μm.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Injection, Staining

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A and B) Injection of Sirt3 siRNA increased intracellular ROS levels, as estimated by CM-H2DCFDA fluorescence intensity. This increase was abolished by NAC (A) and stigmatellin (B), but was only partially decreased by apocynin (B). Embryos were injected with control or Sirt3 siRNA at the pronuclear stage and were cultured with or without NAC for 72 hours. To identify the major origin of increased intracellular ROS, embryos were treated with apocynin or stigmatellin for 30 minutes before CM-H2DCFDA staining. Quantitative data of fluorescence intensity, obtained using ImageJ, were standardized by dividing each value by the average value of the control group in each experiment. Data are derived from 3 (A) or 4 (B) independent experiments. Statistical assessments were performed by applying Games-Howell test. *P < 0.05; **P < 0.001. (C and D) Representative images of CM-H2DCFDA fluorescence in embryos analyzed in A and B, respectively. Scale bars: 100 μm.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Injection, Fluorescence, Cell Culture, Staining, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A) Intracytoplasmic injection of both stealth Sirt3 siRNAs led to decreased blastocyst formation rate in preimplantation embryos. Data are derived from 4 independent experiments. (B) Sirt3 siRNA–induced decrease in blastocyst formation rate was suppressed by treatment with NAC. Data are derived from 6 independent experiments. Statistical assessments were performed by applying Ryan’s multiple-comparison test. *P < 0.05; **P < 0.001.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Injection, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A) Intracytoplasmic injection of Sirt3 siRNA did not affect 2-cell and blastocyst formation rates in low-oxygen (5% O2) conditions. Data are derived from 4 independent experiments. (B) Effects of siRNA-mediated Sirt3 knockdown on intracellular ROS levels, as estimated by CM-H2DCFDA fluorescence intensity, in 20% and 5% O2 conditions. Sirt3 knockdown–induced ROS increases were canceled in 5% O2 conditions. Data are derived from 3 independent experiments. Statistical assessments were performed by applying Games-Howell test. *P < 0.05. (C) Representative images of CM-H2DCFDA fluorescence in embryos analyzed in B. Scale bar: 100 μm.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Injection, Derivative Assay, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: Effect of maternal Sirt3 genotype on IVF rate

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: Effect of maternal Sirt3 genotype on blastocyst formation rate after IVF

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A) After Sr2+ activation for 4.5 hours, eggs that formed 2 pronuclei were injected with control or Sirt3 siRNA, and the rates of 2-cell and blastocyst formation were evaluated. (B) Eggs collected from wild-type and Sirt3–/– female mice underwent parthenogenetic activation. Data are derived from 4 (A) or 2 (B) independent experiments. Statistical assessments were performed by applying Fisher’s exact test. **P < 0.001.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Activation Assay, Injection, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A and B) Implantation rates (A) and full-term survival rates (B) of embryos injected with Sirt3 or control siRNA and transferred into pseudopregnant mice at the 2-cell or morula/blastocyst stage. Implantation sites and viability of fetuses were inspected by cesarean section 18 days after transfer. Implantation rate was estimated by the number of implantation sites; full-term survival rate was assessed by dividing the number of live pups by the number of implantation sites. Data are derived from 5 (2-cell embryo transfer) or 4 (morula/blastocyst transfer) independent experiments. Statistical assessments were performed by applying Ryan’s multiple-comparison test. *P < 0.005; **P < 0.001.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Injection, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A) p53 and p21 were upregulated in Sirt3–/– embryos as in H2O2-treated wild-type embryos. Nanog expression was decreased in Sirt3–/– embryos, and the decrease was enhanced by H2O2 stimulus. (B) Effects of Sirt3 knockdown and treatment with NAC on the expression of p53 and its downstream genes. In Sirt3 siRNA–injected embryos, p21 expression was upregulated, whereas Nanog expression was downregulated. These effects were blocked by NAC. (C) Western blotting analysis showing increased p53 protein levels in Sirt3-knockdown embryos at the morula stage. Signals for acetylated histone H3 (Ac-H3) served as an internal control. (D) Effects of p53 knockdown on Sirt3 siRNA–induced changes in the expression of genes downstream of p53. Sirt3 siRNA–induced p21 upregulation and Nanog downregulation were blocked by siRNA-mediated p53 knockdown. Ppia expression served as an internal control in A, B, and D. (E) Effects of p53 knockdown on preimplantation developmental arrest in Sirt3-knockdown embryos. The rate of blastocyst formation was significantly improved by coinjection with p53 siRNA. Data are derived from 4 independent experiments. Statistical assessments were performed by applying Ryan’s multiple-comparison test. *P < 0.05; **P < 0.001.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Expressing, Injection, Western Blot, Derivative Assay

Journal: Journal of Clinical Medicine

Article Title: Impact of Short-Term Hypoxia on Sirtuins as Regulatory Elements in HUVECs

doi: 10.3390/jcm9082604

Figure Lengend Snippet: Relative gene expression of sirtuins in human umbilical vein endothelial cells (HUVECs). Expression of SIRT1 ( A ), SIRT3 ( B ), and SIRT4 ( C ) in HUVECs. Controls under the normoxic conditions were compared with cells incubated under hypoxia for 10, 60, and 120 min. Shown are the median, upper, and lower quartile, and the 5% and 95% percentile. Each group had a sample size of n = 3. Significance: * = p < 0.05.

Article Snippet: Primary antibodies used were SIRT1 rabbit monoclonal (MerckMillipore, Merck KGaA, Darmstadt, Germany) (MW: ~130 kDa), SIRT3 rabbit polyclonal (MerckMillipore, Merck KGaA, Darmstadt, Germany) (MW: ~46 kDa); SIRT4 rabbit polyclonal (Santa Cruz Biotechnolgy, Inc., Dallas, USA) (MW: ~39 kDa); α-Tubulin rabbit polyclonal (Cell Signaling Technology, Inc., Danvers, USA) (MW: ~52 kDa).

Techniques: Expressing, Incubation

Journal: Journal of Clinical Medicine

Article Title: Impact of Short-Term Hypoxia on Sirtuins as Regulatory Elements in HUVECs

doi: 10.3390/jcm9082604

Figure Lengend Snippet: Western blot analysis and relative protein content of sirtuins in HUVECs. The relative protein content of SIRT1 ( A ), SIRT3 ( B ), and SIRT4 ( C ) in HUVECs. Controls under the normoxic conditions were compared with cells incubated under hypoxia for 10, 60, and 120 min. Shown are the median, upper, and lower quartile, and the 5% and 95% percentile. Each group had a sample size of n = 3. Significance: * p < 0.05 and ** p < 0.005. Representative Western blot analysis ( D ) of cells incubated under normoxia (21% O 2 ) and hypoxia (2% O 2 ) for 10, 60, and 120 min. α-Tubulin ~52kDa; SIRT1 ~110 kDa; SIRT3 ~44 kDa; SIRT4 ~39 kDa.

Article Snippet: Primary antibodies used were SIRT1 rabbit monoclonal (MerckMillipore, Merck KGaA, Darmstadt, Germany) (MW: ~130 kDa), SIRT3 rabbit polyclonal (MerckMillipore, Merck KGaA, Darmstadt, Germany) (MW: ~46 kDa); SIRT4 rabbit polyclonal (Santa Cruz Biotechnolgy, Inc., Dallas, USA) (MW: ~39 kDa); α-Tubulin rabbit polyclonal (Cell Signaling Technology, Inc., Danvers, USA) (MW: ~52 kDa).

Techniques: Western Blot, Incubation

Journal: Journal of Clinical Medicine

Article Title: Impact of Short-Term Hypoxia on Sirtuins as Regulatory Elements in HUVECs

doi: 10.3390/jcm9082604

Figure Lengend Snippet: Relative sirtuin enzyme capacity in the HUVECs. Relative maximum activity of SIRT1 ( A ) and SIRT3 ( B ) in the HUVECs. Normoxic controls were compared with cells incubated under hypoxia for 10, 60, and 120 min. Shown are the median, upper, and lower quartile, and the 5% and 95% percentile. Each group had a sample size of n = 3. Normoxic SIRT1 enzyme capacity averaged 1.8 U/10,000 cells in the normoxic HUVECs. The normoxic enzyme activity of SIRT3, averaged 9.5 U/µg protein.

Article Snippet: Primary antibodies used were SIRT1 rabbit monoclonal (MerckMillipore, Merck KGaA, Darmstadt, Germany) (MW: ~130 kDa), SIRT3 rabbit polyclonal (MerckMillipore, Merck KGaA, Darmstadt, Germany) (MW: ~46 kDa); SIRT4 rabbit polyclonal (Santa Cruz Biotechnolgy, Inc., Dallas, USA) (MW: ~39 kDa); α-Tubulin rabbit polyclonal (Cell Signaling Technology, Inc., Danvers, USA) (MW: ~52 kDa).

Techniques: Activity Assay, Incubation

Journal: PLoS ONE

Article Title: Changes in Muscle Cell Metabolism and Mechanotransduction Are Associated with Myopathic Phenotype in a Mouse Model of Collagen VI Deficiency

doi: 10.1371/journal.pone.0056716

Figure Lengend Snippet: Histograms and representative immunoblot images of FASN (panel A), PPARγ, α and β (panel C), PGC1-α (panel D), SIRT1 and SIRT3 (panel E) in gastrocnemius, tibialis anterior and diaphragm from Col6a1 −/− and wild-type mice (n = 4; mean ± S.D.; Student’s T-test). Panel B: electron microscopy image showing lipid accumulation in the diaphragm of Col6a1 −/− mice. Black arrows show lipid droplets.

Article Snippet: Blots were incubated with rabbit or goat polyclonal primary antibodies (Cell Signaling Technology and Santa Cruz Biotechnology) as follows: anti-TN-C (1∶500), anti-ROCK1 (1∶500), anti PI3K (1∶1000), anti FAK (1∶1000), anti-PPARγ (1∶1000), anti-PPARα (1∶1000), anti-PPARβ (1∶1000), anti-Sirt1 (1∶1000), anti-Sirt3 (1∶1000), anti-FASN (1∶1000), anti-IDH1 (1∶1000), anti-IDH2 (1∶1000) and anti β-tubulin (1∶1000).

Techniques: Western Blot, Electron Microscopy