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

Journal: Autophagy

Article Title: Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein.

doi: 10.1080/15548627.2020.1841953

Figure Lengend Snippet: Figure 4. Biophysical validation reveals VDAC1 as a new protein target of Sert. (A) The DARTS assay for target validation. VDAC1 protein stability was increased upon Sert (100 μM) treatment in HUVECs lysates. Pronase treatment was conducted for 5, 10, and 20 min. The graph plots the quantification data is shown in (A). n = 3, *p < 0.05, **p < 0.01. (B) The DARTS assay demonstrated the dose-dependent binding of Sert to VDAC1. Treatment with pronase (5 μg/mL) was conducted for 10 min. The graph plots the quantification data is shown in (B). n = 3, *p < 0.05, **p < 0.01. (C) In silico docking model of Sert interacting with VDAC1 (human VDAC1, RCSB PDB ID: 2JK4). Sert bound to the VDAC1 between α-helix and β-sheet in the most stable pose and binding motifs were depicted with several high- affinity interactions between Sert and the VDAC1 pocket. Ligands are shown as gray sticks in displayed hydrogen bond surfaces. Bonds are shown as dashed lines color-coded as follows: hydrophobic interactions in orange, electrostatic interaction in purple, and hydrogen bonds in green and sky blue.

Article Snippet: Recombinant VDAC1 plasmid transfection analysis For transient overexpression of VDAC1, MYC-VDAC1 (Origene, RC209949) was purchased.

Techniques: Biomarker Discovery, Binding Assay, In Silico

Journal: Autophagy

Article Title: Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein.

doi: 10.1080/15548627.2020.1841953

Figure Lengend Snippet: Figure 5. Biological validation using vdac1-knockout cells reveals that VDAC1 is a biological relevant protein target of Sert. (A) Western blot analysis of the levels of VDAC1, p-MTOR, MTOR, p-AMPK, AMPK, and ACTB in cells after each compound treatment for 30 min in WT MEFs or vdac1-knockout MEFs. The graph plots the quantification data is shown in (A). n = 3, *p < 0.05, **p < 0.01. (B) MAP1LC3 positive puncta (green) were increased by Sert (5 μM) treatment in WT MEFs, whereas vdac1-knockout MEFs treated with Sert did not induce autophagic vesicles formation. The cells were treated with Sert (5 μM) or Rapa (10 μM) for 24 h in HUVECs. And then, the cells were harvested, followed by treatment with MAP1LC3B (green) antibody for 1 h. Nuclei were stained with DAPI. Images were obtained from confocal microscopy. Quantification of data is shown in (B). Values are means ± SEM; n > 10 cells, ***p < 0.001; scale bar: 10 μm. (C) Autophagic flux evaluation in WT MEFs or vdac1-knockout MEFs using mCherry-GFP MAP1LC3 in the presence of each compound. Representative images of merged channels are shown; scale bar: 10 μm. Pearson coefficient for the colocalization analysis is shown. Quantification of data is shown in (C). Values are means ± SEM; n > 10 cells, **p < 0.01, ***p < 0.001. (D) Effect of Sert treatment on the proliferation of WT MEFs and vdac1-knockout MEFs. All cells were treated with Sert (0–5 μM) for 72 h, and cell growth was measured using an MTT colorimetric assay. N = 3, Values are means ± SEM. **p < 0.01, ***p < 0.001.

Article Snippet: Recombinant VDAC1 plasmid transfection analysis For transient overexpression of VDAC1, MYC-VDAC1 (Origene, RC209949) was purchased.

Techniques: Biomarker Discovery, Knock-Out, Western Blot, Staining, Confocal Microscopy, Colorimetric Assay

Journal: Autophagy

Article Title: Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein.

doi: 10.1080/15548627.2020.1841953

Figure Lengend Snippet: Figure 6. Validation of the binding site of Sert on VDAC1 (A) For DARTS assay, vdac1−/- MEF cells were transfected with WT MYC-VDAC1, MYC-VDAC1H184P or MYC- VDAC1S196A vectors at 1500 nM each for 48 h. And then, the cells were harvested, lysed, followed by treatment with (Sert, 100 μM) treatment. Pronase treatment (5 μg/mL) was conducted for 10 min. The graph plots the quantification data is shown in (A). n = 3, *p < 0.05. (B) VDAC1−/- MEF cells were transfected with WT MYC- VDAC1, MYC-VDAC1H184P or MYC-VDAC1S196A vectors at 200 nM each for 24 h. Then, the cells were treated with Sert (5 μM) for an additional 24 h. And then, the cells were harvested, followed by treatment with MYC (red) and MAP1LC3B (green) antibody for 3 h. Nuclei were stained with DAPI. Images were obtained from confocal microscopy. Quantification of data is shown in (B). Values are means ± SEM; n > 10 cells, *p < 0.05, **p < 0.01, ***p < 0.001; scale bar: 10 μm.

Article Snippet: Recombinant VDAC1 plasmid transfection analysis For transient overexpression of VDAC1, MYC-VDAC1 (Origene, RC209949) was purchased.

Techniques: Biomarker Discovery, Binding Assay, Transfection, Staining, Confocal Microscopy

Journal: Autophagy

Article Title: Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein.

doi: 10.1080/15548627.2020.1841953

Figure Lengend Snippet: Figure 9. VDAC1 is a biologically relevant target protein of Sert-induced biological activity on tauopathy. (A) Western blot analysis of the level of VDAC1, p-MAPT, MAPT, Cleaved MAPT, MAP1LC3, and ACTB in inducible MAPT cells after transfection with si-VDAC1 (100 nM) for 48 h. The graph plots the quantification data is shown in (A). n = 3, *p < 0.05, **p < 0.01, ***p < 0.001. (B) Degradation of MAPT oligomers was visualized in MAPT-BiFC cells when cells were transfected with si- VDAC1 (100 nM) for 48 h. Scale bar: 20 μm. Quantification of data is shown in (B). Values are means ± SEM; n > 20 cells. ***p < 0.001. (C) Schematic summary of VDAC1-mediated autophagy modulation upon Sert treatment.

Article Snippet: Recombinant VDAC1 plasmid transfection analysis For transient overexpression of VDAC1, MYC-VDAC1 (Origene, RC209949) was purchased.

Techniques: Activity Assay, Western Blot, Transfection