Journal: bioRxiv

Article Title: Mitochondria – insulin granule crosstalk controls the early stages of granule maturation

doi: 10.64898/2026.02.23.707428

Figure Lengend Snippet: VNUT and VDAC associate with newly formed insulin granules (A) Confocal microscopy images of a MIN6 cell immunostained against insulin (green) and VNUT (magenta). (B) Confocal microscopy images of a MIN6 cell immunostained against insulin (green), VDAC (yellow) and VNUT (red). (C) Quantification of the colocalization between VDAC and NPY-Halo. A pulse-chase protocol for Halo labeling was employed where pre-existing granules were labeled with JFX549 (old) and newly synthesized granules with JFX650 (old) (means ± SEM; n = 25 cells from three experiments; Mann-Whitney U-test; *p < 0.05). (D) Confocal microscopy images of mouse islets expressing the AdRIP-proCpepRUSH 30, 60 and 120 min post-biotin addition (200 μM; cyan) and immunostained against VDAC (yellow) and VNUT (red). (E) Confocal microscopy images from a mouse islet showing PLA signal between VNUT and VDAC1 (magenta) and insulin immunoreactivity (cyan). The white region is expanded below. (F) Average number of PLA puncta (using antibodies against VNUT and VDAC1 or VNUT and VDAC2) per insulin-positive cell (single confocal plane; means ± SEM, n = 4 experiments).

Article Snippet: The following antibodies and dyes were used in the study: Rab3 monoclonal (catalog no. 107 111, Synaptic Systems, host: mouse, 1:500), TOM20 polyclonal (catalog no. 11802-1-AP, Proteintech, host: Rabbit, 1:400), insulin polyclonal (catalog no. A0564, Dako, host: guinea pig, 1:1000), VDAC (catalog no. MA533205, host: rabbit, 1:200, Invitrogen), VDAC1 (catalog no. 66345-1-lg, Proteintech, host: mouse, 1:200), VDAC2 (catalog no. 66388-1-lg, Proteintech, host: mouse, 1:200), VNUT polyclonal (catalog no. ABN83, Merck, host: guinea pig, 1:200).

Techniques: Confocal Microscopy, Pulse Chase, Labeling, Synthesized, MANN-WHITNEY, Expressing

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

Journal: Autophagy

Article Title: CASP9 (caspase 9) is essential for autophagosome maturation through regulation of mitochondrial homeostasis.

doi: 10.1080/15548627.2019.1695398

Figure Lengend Snippet: Figure 11. Mitochondrial fusion-fission proteins are affected by CASP9 KO. (A) Western blotting analysis of mitochondrial fusion and fission proteins in sgCon and sgCASP9 cells. Graphs, quantification of DNM1L, MFN1 and MFN2 normalized to ACTB or p-DNM1L S616 and S637 normalized to total DNM1L (n = 4). (B) Mitochondrial morphology after expression of DN-DNM1L. HeLa cells transfected with IRES-mCherry (EV) or DN-DNM1L-IRES-mCherry (DN-DNM1L) were stained with MitoTracker Green (200 nM, 20 min), and analyzed using live-imaging confocal microscopy. Scale bar: 20 μm (5 μm for magnified images). (C) Western blotting analysis of GABARAP1L, L2 and MAP1LC3B lipidation transfected with IRES-mCherry (EV) or DN-DNM1L-IRES-mCherry (DN-DNM1L) after AA starvation for 1 h without or with BafA1 (100 nM) in HeLa cells (n = 3). (D) Western blotting analysis of mitochondrial localization of cleaved CASP9 in HeLa cells after AA starvation for the indicated time periods. Cyto and Mito represent cytoplasmic and mitochondria fractions, respectively. TUBB and VDAC were used as loading control of each fraction. (E) CASP9 activity in HeLa cells after AA starvation without or with Mito-TEMPO (10 μM) (n = 5). (F) Western blotting analysis of cleaved CASP9 after AA starvation for 1 h with Mito-TEMPO at the indicated concentrations (n = 4). Mito-TEMPO was added 2 h before medium change to EBSS. S. E., short exposure; L. E., long exposure. *P < 0.05, **P, ##P < 0.01, ***P < 0.001; ns, not significant.

Article Snippet: Antibodies against the following proteins were used: MAP1LC3B (NB100-2220), ATG5 (NB110-53818), and APAF1 (NBP1-76999) from Novus Biologicals; GABARAPL1 (11010-1-AP) and GABARAPL2 (18724-1-AP) from Proteintech; FLAG (F3165) from SigmaAldrich; GABARAP (13733S), p-MTOR S2448 (5536), MTOR (2972S), p-ULK1 S757 (14202), ULK1 (8054S), BECN1 (3738S), ATG3 (3415), ATG7 (8558), ATG16L1 (8089), pro-CASP3 (9662), cleaved CASP3 (9661), CASP9 (9508), cleaved CASP9 (9595 for human, 9507 for rat), PARP1 (9542), TUBB (2146), VDAC (4866), p-DNM1L S616 (4494), and MFN2 (9482) from Cell Signaling Technology; p-BECN1 S15 (254515) from Abbiotec; p-DNM1L S637 (orb 127984) from Biorbyt; RAB7 (sc376362), DNM1L (sc-32898), MFN1 (sc-166644) and horseradish peroxidase-conjugated ACTB/β-actin (sc-47778) from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Transfection, Staining, Imaging, Confocal Microscopy, Control, Activity Assay

Journal: Molecular nutrition & food research

Article Title: Lack of β, β-carotene -9’, 10’-oxygenase 2 leads to hepatic mitochondrial dysfunction and cellular oxidative stress in mice

doi: 10.1002/mnfr.201600576

Figure Lengend Snippet: Functional categorization of proteins differentially expressed in hepatic mitochondria of BCO2 knockout (KO) vs. 129S6 wild type (WT) mice identified by spectrum counting. Values are means, n=3 mice/group with 4 technical replicates/mouse.

Article Snippet: Antibodies against fatty acid synthase (FASN) (catalog # 10624-2-Ap), ATP synthase α subunit 1 (ATP5A1) (catalog # 14676-1-AP), citrate synthase (catalog # 16131-1-AP), hydroxyacyl-coenzyme A dehydrogenase (HADH) (catalog # 19828-1-AP), succinate dehydrogenase α (SDHA) (catalog # , 14865-1-AP), and nicotinamide nucleotide transhydrogenase (NNT) (catalog # 13442-2-AP) were purchased from ProteinTech Group (Chicago, IL, USA); Antibodies against lysosome-associated membrane protein 1 (LAMP1) (catalog # sc-20011), catalase (catalog # sc-50508), NNT (catalog # sc-163154), glutathione reductase (GSR) (catalog # sc-133245) were purchased from Santa Cruz Biotech (Dallas, TX, USA); peroxisome proliferator-activated receptor α (PPARα) antibody (catalog # 101710) was purchased from Cayman (Ann Arbor, MI, USA); Antibodies against β-actin (catalog # 4967), phosphor-Thr172-AMP-activated protein kinase α (pT172-AMPKα) (catalog # 2535), and AMPKα (catalog # 2603) were purchased from Cell Signaling Technology (Danvers, MA, USA); voltage-dependent anion-selective channel protein 1 (VDAC1) antibody (catalog # PA1780) was purchased from Boster Biosciences (Pleasanton, CA, USA).

Techniques: Functional Assay, Knock-Out, Membrane