Journal: Cell Reports Medicine

Article Title: Urolithin A improves muscle strength, exercise performance, and biomarkers of mitochondrial health in a randomized trial in middle-aged adults

doi: 10.1016/j.xcrm.2022.100633

Figure Lengend Snippet: Urolithin A confers a proteomic signature of improved mitochondrial metabolism and mitophagy in human skeletal muscle (A) Venn diagram summarizing pathway enrichment analysis results from proteomics data in vastus lateralis skeletal muscle. Data represent upregulated pathways with an adjusted p value <0.1 in subjects treated with placebo or with UA at 500 and 1,000 mg for 4 months compared with baseline. (B and C) Dot plots showing top enriched pathways (WikiPathways 2019 Human), ranked by protein ratio, in the UA 500- (B) and UA 1,000 mg (C) groups from (A). Dot color and size indicate adjusted p value and protein count, respectively. Significant pathways for the placebo group were filtered out to identify treatment-specific pathways. (D) Western blot analysis of protein lysates from vastus lateralis skeletal-muscle biopsies in subjects treated as above. For each subject, both baseline (Pre) and end-of-the-treatment (Post) samples were run, and membranes were probed for phospho-Parkin, total Parkin, and the mitochondrial proteins ATP5A (belonging to the OXPHOS complex V), UQCRC2 (complex IV), SDHB (complex II), and NDUFB8 (complex I). Tubulin and VDAC were included as markers of total and mitochondrial protein abundance, respectively. Dashed line separates samples from individual subjects. (n = 6 Pre and Post, biologically independent samples). (E) Quantification of phospho-Parkin over Tubulin protein intensity from western blots (WBs) in (D) (n = 6). Two-sided, paired t-test. (F) Quantification of NDUFB8 (left) and SDHB (right) protein intensity, normalized over VDAC from WBs in (D) (n = 6). ∗p < 0.05; ∗∗p < 0.01; two-sided, paired t test.

Article Snippet: The following primary antibodies were incubated overnight diluted in blocking buffer: UBE2N (SantaCruz, sc-376470, 1:3000), Tubulin (Proteintech, 10004185, 1:3000), phospho-Parkin S65 (Cell Signaling, #36866, 1:1000), Parkin (Cell Signaling, #4211, 1:1000), OXPHOS Antibody Cocktail (Abcam, ab110413, 1:2000), VDAC (Proteintech, 10866-1-AP, 1:1000).

Techniques: Western Blot, Quantitative Proteomics

Journal: Cell Reports Medicine

Article Title: Urolithin A improves muscle strength, exercise performance, and biomarkers of mitochondrial health in a randomized trial in middle-aged adults

doi: 10.1016/j.xcrm.2022.100633

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were incubated overnight diluted in blocking buffer: UBE2N (SantaCruz, sc-376470, 1:3000), Tubulin (Proteintech, 10004185, 1:3000), phospho-Parkin S65 (Cell Signaling, #36866, 1:1000), Parkin (Cell Signaling, #4211, 1:1000), OXPHOS Antibody Cocktail (Abcam, ab110413, 1:2000), VDAC (Proteintech, 10866-1-AP, 1:1000).

Techniques: Recombinant, RNA Extraction, Software, Protease Inhibitor

Journal: Aging Cell

Article Title: The ultrastructural and proteomic analysis of mitochondria‐associated endoplasmic reticulum membrane in the midbrain of a Parkinson's disease mouse model

doi: 10.1111/acel.14436

Figure Lengend Snippet: Initial proteomic parsing of midbrain MAM fractions in controls and MPTP‐treated mice. (a) Subcellular fractions extracted from midbrain tissues were validated by specific organelle protein markers. Calnexin, Calreticulin, and Sigma‐1R were considered as consensus proteins for ER and MAM. VDAC1 and Cox IV were applied for mitochondria and MAM markers, and GAPDH was to prove cytosolic fractions. (b) Venn graph of identified proteins in each group ( n = 5 per group) and consensus MAM proteins. (c, d) Sample distribution plots by PCA (c) and PLS‐DA analysis (d) in MAM proteomics between controls and MPTP‐treated mice.

Article Snippet: Primary antibodies used in this study included that anti‐tyrosine hydroxylase (TH) rabbit mAb (1:2000, Cat. 58844, Cell Signaling Technology), anti‐glial fibrillary acidic portein (GFAP) rabbit mAb (1:2000, Cat. 80788, Cell Signaling Technology), anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) rabbit mAb (1:2000, Cat. 5174, Cell Signaling Technology), anti‐cytochrome‐c‐oxidase subunit 4 (Cox IV) rabbit Ab (1:2000, Cat. 5844, Cell Signaling Technology), anti‐sigma‐1 receptor (Sig‐1R) rabbit mAb (1:2000, Cat. 61994, Cell Signaling Technology), anti‐phosphatase and tensin homolog (PTEN) rabbit mAb (1:2000, Cat. 9188, Cell Signaling Technology), anti‐calreticulin rabbit mAb (1:2000, Cat. 92516, Abcam), anti‐calnexin (CNX) rabbit pAb (1:2000, Cat. ADI‐SPA‐860, Enzo Life Sciences), anti‐voltage dependent anion channel 1 (VDAC1) rabbit pAb (1:2000, Cat. 55259‐1‐AP, Proteintech), anti‐leucine‐rich repeat kinase 2 (LRRK2) rabbit mAb (1:1000, Cat. R380823, Zen‐bioscience), anti‐excitatory amino acid transporter 2 (EAAT2) rabbit mAb (1:1000, Cat. R381853, Zen‐bioscience), anti‐tropomyosin‐1 (TPM1) rabbit mAb (1:1000, Cat. R383145, Zen‐bioscience), and anti‐coatomer protein complex subunit zeta 1 (COPZ1) rabbit pAb (1:1000, Cat. 824631, Zen‐bioscience).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Integrated Multilayer Omics Reveals the Underlying Mechanisms in Xylazine-Related Heart Injury in Rats

doi: 10.3390/ijms26178532

Figure Lengend Snippet: Validation of related proteins in the fructose and mannose metabolism signaling pathway and cholesterol metabolism signaling pathway. ( A ) Western blot image of VDAC3. ( B ) Quantification of VDAC3. ( C ) Western blot image of PFKM. ( D ) Quantification of PFKM. * p < 0.05 compared with the control group.

Article Snippet: The membranes were incubated in 5% skimmed milk for 2 h, and then in diluted primary antibodies against PFKM (1:2000, 55028-1-AP, Proteintech, Rosemont, IL, USA); VDAC3(1:500, 55260-1-AP, Proteintech), GAPDH (1:10,000, 60004-1-Ig, Proteintech), tubulin (1:10,000, GB15140-100, Servicebio) at 4 °C 12 h. They were incubated in Goat Anti-Rabbit IgG H&L/HRP (1:10,000, GB23303, Servicebio) or Goat Anti-Mouse lgG H&L/HRP (1:10,000, GB23301, Servicebio) for 1 h. A chemiluminescence instrument was used to image the bands and Image J v1.8.0.345 was used to calculate the gray values.

Techniques: Biomarker Discovery, Western Blot, Control