Journal: bioRxiv

Article Title: SIRT3 deficiency decreases oxidative-metabolism capacity but increases lifespan under caloric restriction

doi: 10.1101/2022.05.09.491205

Figure Lengend Snippet: A) Immunoblot of pan-acetylation, normalized to VDAC, for liver mitochondrial enrichment from 25-month-old treatment groups. Data were analyzed by two-way ANOVA followed by multiple comparisons test. p value reported for each comparison is corrected by Tukey’s test. Results are plotted as mean ± SEM. *: p≤0.05. B) Percentage of significantly changed acetyl-lysine residues that show increased stoichiometry due to Sirt3 -/- , calculated by (the number of acetyl-lysine sites showing increased stoichiometry) / (the number of significantly changed acetyl-lysine sites, p<0.05) x100%. C) Heat map of significantly changed lysine sites (p<0.1) that are response to loss of SIRT3. Plotted sites are significantly changed (p<0.1) in either Sirt3 -/- CD vs. WTCD or Sirt3 -/- CR vs. WTCR comparison. Values are colored based on relative acetylation stoichiometry, normalized to the median value of each sites in all four groups, scaling ranging from -0.8 to 0.8 (x100%). D) Functional cluster analysis of KEGG pathways (DAVID 6.8). Significantly enriched (-log10(p value) >1.5) pathways are indicated, with Sirt3 -/- CD vs. WTCD in orange and Sirt3 -/- CR vs. WTCR in blue. E) Acetylation sites in FAO and BACC metabolism, TCA cycle, and ETC that displayed larger than 5% stoichiometry (p<0.1) for Sirt3 -/- CD vs. WTCD (orange colored) and Sirt3 -/- CR vs. WTCR comparison (blue colored).

Article Snippet: Western blot primary antibodies used include: SIRT3 (#5490, CST, 1:1000), Acetylated-Lysine (#9681, CST, 1:1000), VDAC (75-204, NeuroMab, 1:1250).

Techniques: Western Blot, Comparison, Functional Assay

Journal: Biomedical Journal of Scientific & Technical Research

Article Title: "Age-Related Variations of the Brain Mitochondrial Permeability Transition Pore and Complex I in Response to Hypoxia"

doi: 10.26717/bjstr.2023.51.008169

Figure Lengend Snippet: Figure 1: Protein levels of CypD and NDUFS2 relative to VDAC1. (a) Representative blots and quantification of relative CypD levels (n=4). (b) Representative blots and quantification of relative NDUFS2 levels (n=5). *- statistically significantly different compared to 24–26 months group.

Article Snippet: The primary antibodies were: NDUFS2 antibody (NBP230413, Novus biologicals) diluted 1:1000 in blocking buffer (5% BSA in TBST); Cyclophilin-F Antibody (JM71-39, Novus biologicals) diluted 1:2000 in blocking buffer; and VDAC1 Antibody (SA93-03, Novus biologicals) diluted 1:2000 in blocking buffer.

Techniques:

Journal: Scientific Reports

Article Title: The transcriptome of mouse central nervous system myelin

doi: 10.1038/srep25828

Figure Lengend Snippet: ( A ) Scheme illustrating the biochemical purification of a light-weight membrane fraction enriched for myelin by homogenizing mouse brains in 0.32 M sucrose, sequential sucrose density gradient centrifugation, and osmotic shocks. Myelin accumulates at the interface between 0.32 M and 0.85 M sucrose. ( B ) Immunoblot analysis of myelin-enriched fractions and equal amounts of brain lysate to compare the abundance of marker proteins for compact myelin (PLP/DM20, MBP), non-compact myelin (CNP, SIRT2), the oligodendroglial nuclear/cytoplasmic compartment (OLIG2), astrocytes (GFAP), microglia (AIF1/IBA1), neuronal plasma membrane (GPM6A), axonal microtubules (TUBB3/TUJ1), and mitochondria (VDAC). Blot represents three biological replicates (male c57Bl6/N mice, age P75). Note that myelin markers were enriched in purified myelin while markers of other cellular sources were reduced. ( C ) Heatmap displaying reverse CT values from qRT-PCRs for three markers each specific for myelin, migroglia, neurons and astrocytes performed on myelin biochemically purified from the brains of 4 individual mice (M1–4) compared to the respective brain lysates (BL1–4) at six month of age.

Article Snippet: Antibodies were specific for PLP/DM20 (A431 , 1:5000), MBP (Dako A0623, 1:500), CNP (Sigma C 5922, 1:1000), SIRT2 (Abcam ab67299, 1:500), OLIG2 (DF308 , 1:200, kindly provided by J. Alberta and C. Stiles, Boston, MA, USA) GFAP (Novocastra NCL-GFAP-GA5, 1:500), AIF (also termed IBA1; Abcam ab107159, 1:500), GPM6A (#24924 , 1:1000), TUBB3 (also termed TUJ1; Covance MMS-435P, 1:1000), and VDAC (Rockland 600-401-882, 1:2000).

Techniques: Purification, Membrane, Gradient Centrifugation, Western Blot, Marker, Clinical Proteomics