Journal: Proteomes

Article Title: Calcium Homeostasis and Muscle Energy Metabolism Are Modified in HspB1-Null Mice

doi: 10.3390/proteomes4020017

Figure Lengend Snippet: Suppliers and conditions for each antibody used in this study.

Article Snippet: HspA9 , Stress-70 protein, mitochondrial , Monoclonal , R & D Systems: MAB3584 , 1/200.

Techniques:

Journal: Cells

Article Title: Steroidogenesis Upregulation through Mitochondria-Associated Endoplasmic Reticulum Membranes and Mitochondrial Dynamics in Rat Testes: The Role of D-Aspartate

doi: 10.3390/cells13060523

Figure Lengend Snippet: List of all the used antibodies.

Article Snippet: GRP75 , 75 , 1:1000 , - , Cell Signaling Technology, Danvers, MA, USA #2816.

Techniques: Molecular Weight, Plasmid Preparation

Journal: Cells

Article Title: Steroidogenesis Upregulation through Mitochondria-Associated Endoplasmic Reticulum Membranes and Mitochondrial Dynamics in Rat Testes: The Role of D-Aspartate

doi: 10.3390/cells13060523

Figure Lengend Snippet: Analysis of calcium signaling ( A ) and ER stress ( B ) in control and D-Asp-treated rats. ( A ) Western blot analysis of VDAC (32 kDa) and GRP75 (75 kDa) protein levels in the testes of animals treated with D-Asp for 15 days compared to controls. ( B ) Western blot analysis of GRP78 (78 kDa) protein levels in rat testes of D-Asp group compared to controls. The expression levels of VDAC, GRP75, and GRP78 were quantified using ImageJ and normalized to β-actin (42 kDa). All values are expressed as the mean ± SD of 5 animals in each group. D-Asp vs. Control (C): * p < 0.05.

Article Snippet: GRP75 , 75 , 1:1000 , - , Cell Signaling Technology, Danvers, MA, USA #2816.

Techniques: Control, Western Blot, Expressing

Journal: Cell proliferation

Article Title: SIRT1 Alleviates Oxidative Stress-Induced Mitochondrial Dysfunction and Mitochondria-Associated Membrane Dysregulation in Stress Urinary Incontinence.

doi: 10.1111/cpr.70009

Figure Lengend Snippet: FIGURE 8 | The effects of SIRT1 upregulation on structural and functional alterations of MAMs and calcium homeostasis in L929 cells under oxi- dative stress. (A) Immunoblot analyses of PERK and GRP78 in L929 cells. Quantification represents the levels of the indicated proteins normalised to β-Actin. (B) Representative TEM images of mitochondrial ultrastructure of L929 cells. Blue arrows indicated MAMs. Quantitative analysis of MAM parameters, including MAM distance and length to mitochondrion in TEM images. (C) Immunoblot analyses of SERCA2, GRP75, MCU and VDAC1 in L929 cells. Quantification represents the levels of the indicated proteins normalised to β-Actin. (D) Co-localization analysis of immunofluores- cence images of IP3R (green) and MitoTracker (red) in different groups of L929 cells. (E) Co-localization fluorescence imaging was conducted with MitoTracker, demonstrating that Rhod-2 AM is predominantly an indicator of mitochondrial Ca2+ levels. (F) Concentration of mitochondrial calcium ions. (G) Representative flow plots of mitochondrial ROS production in L929 cells measured by flow cytometry. Data are expressed as mean ± SD. *p < 0.05 compared with control group, #p < 0.05 compared with H2O2 group.

Article Snippet: After being blocked with 5% skim milk for 1 h, the membranes were incubated overnight at 4°C with primary antibodies against SIRT1 (1:1000, Proteintech Group Inc.), PGC- 1α (1:1000, Proteintech Group Inc.), NRF1 (1:5000 Proteintech Group Inc.), TFAM (1:5000, Proteintech Group Inc.), PINK1 (1:2000, Proteintech Group Inc.), Parkin (1:2000, Proteintech Group Inc.), Collagen I (1:1000, Proteintech Group Inc.), Collagen III (1:1000, Proteintech Group Inc.), Cytochrome c (1:1000, Proteintech Group Inc.), VDAC1 (1:2000, Proteintech Group Inc.), GRP75 (1:5000, Proteintech Group Inc.), FUNDC1 (1:1000, Cell Signalling Technology Inc.), PERK (1:1000, Proteintech Group Inc.), GRP78 (1:3000, Proteintech Group Inc.), SERCA2 (1:5000, Proteintech Group Inc.), MCU (1:3000, Proteintech Group Inc.) and β- actin (1:20,000, Proteintech).

Techniques: Functional Assay, Western Blot, Fluorescence, Imaging, Concentration Assay, Flow Cytometry, Control

Journal: The FASEB Journal

Article Title: The regulation of mitochondrial dynamics in neurite outgrowth by retinoic acid receptor β signaling

doi: 10.1096/fj.201802097r

Figure Lengend Snippet: Figure 4. In the growing neurite, mitochondria interact with GRP75 and the ER following RAR-b activation. A–D) Neurons treated with CD2019 for 3 d [imaging of cell body and neuron (scale bar, 10 mm) (A); zoomed-in imaging of the neurite in a series of z-stack slices (scale bar, 20 mm) (B)] show an increase in colocalization between GRP75 and mitotracker red signals compared with vehicle-treated neurons (C, D). E, F). Treatment of neurons with siRNA for GRP75 negated the effect of neurite elongation of treatment with RAR-b agonist: imaging (green signal is bIII-tubulin; scale bar, 20 mm) (E), quantification (F). G, H) Live imaging of neurons treated with GRP75 siRNA showed that acute treatment with RAR-b agonist (at least 30 min prior to imaging) doubled the number of mitochondria moving in the retrograde direction, whereas the number of anterograde-moving mitochondria is unaltered compared with vehicle-treated neurons: imaging (G), quantification (H). I, J) In similarly treated preparations, ER staining and mitotracker red staining (scale bar, 5 mm) (I) show a solid Pearson’s r 5 0.5 (J), which is not altered significantly with activation of RAR-b. K) The neurite’s more distal 20 mm2 reveals a significant increase in the overlap between ER and mitochondria. At least 5 neurons were quantified from each of 3 slides per condition. N.s., not significant. *P , 0.05, **P , 0.005.

Article Snippet: GRP75 small interfering RNA (siRNA) (sc-35521; Santa Cruz Biotechnology, Dallas, TX, USA) and Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) in OptiMem (2% v/v) were prepared and allowed to stand for 5 min.

Techniques: Activation Assay, Imaging, Staining

Journal: The FASEB Journal

Article Title: The regulation of mitochondrial dynamics in neurite outgrowth by retinoic acid receptor β signaling

doi: 10.1096/fj.201802097r

Figure Lengend Snippet: Figure 5. Model for mitochon- dria recruitment by RAR-b in the growing neurite. Activation of RAR-b in cortical neuronal cultures promotes neurite elon- gation. This process involves, in part, up-regulation of HIF-1a signaling, which promotes mi- tochondria membrane depolar- ization, decreasing oxidative phosphorylation in the cell body, but not neurite tip. Acti- vation of RAR-b also causes a HIF-1a–dependent increase in mitochondria proliferation and increases the speed of mito- chondria movement along the neurite. Following activation of RAR-b, GRP75 mediates mito- chondria binding to the ER in the terminal, anchoring the recruited mitochondria to this region, decreasing the num- ber of mitochondria being transported retrogradely, and effectively increasing the mito- chondrial presence in the grow- ing neurite terminal.

Article Snippet: GRP75 small interfering RNA (siRNA) (sc-35521; Santa Cruz Biotechnology, Dallas, TX, USA) and Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) in OptiMem (2% v/v) were prepared and allowed to stand for 5 min.

Techniques: Activation Assay, Membrane, Phospho-proteomics, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Nogo-B Receptor Directs Mitochondria-Associated Membranes to Regulate Vascular Smooth Muscle Cell Proliferation

doi: 10.3390/ijms20092319

Figure Lengend Snippet: NgBR KD cells show disrupted ER-mitochondria interaction. shControl- and shNgBR-transfected cells were harvested at 48 h post transfection. MAM formation was analyzed by evaluating the expression of MAM-related proteins in ( A ) total cell lysates (FACL-4 and IP 3 R3) and ( B ) crude mitochondrial extracts (FACL-4 and VDAC1); β-actin and COX IV were utilized as the loading controls, respectively. n = 3. ( C ) Cells were incubated with plasmid expressing mCherry fused with a targeting sequence of subunit IV of COX prior to shRNA transfection. PDI was used as an ER marker. Nuclei were stained with Hoechst (blue). Pearson’s correlation coefficient was calculated to measure colocalization of ER and mitochondria on mCherry positive cells. n > 60 cells/group. Scale bar = 10 μm. ( D ) Transmission electron microscopy of negative control (NC; left) and NgBR knockdown KD (right) VSMCs. Arrowheads indicate the MAMs. The minimum ER-mitochondrial distance was quantified. n > 80 ER-mitochondria contact points/group. Scale bar = 500 nm. * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies against proliferating cell nuclear antigen (PCNA; BM0104), VDAC1 (PB0478), GRP75 (PB0668), and IP 3 R1 (PB0223) were obtained from Boster (Pleasanton, CA, USA).

Techniques: Transfection, Expressing, Incubation, Plasmid Preparation, Sequencing, shRNA, Marker, Staining, Transmission Assay, Electron Microscopy, Negative Control, Knockdown

Journal: Translational Oncology

Article Title: Uncovering Differently Expressed Markers and Heterogeneity on Human Pancreatic Cancer

doi: 10.1016/j.tranon.2020.100749

Figure Lengend Snippet: Blind SELEX to identify potential biomarkers on pancreatic cancer. (A) The schematic workflow of blind SELEX is depicted. After untargeted SELEX to identify differently expressed markers on plasma membrane of PANC-1 cells, aptamers are enriched in vitro . Target ligands were retrieved via affinity pull-down assays using biotinylated aptamers. After affinity pull-down assays, MS/MS is used to achieve peptide fingerprinting. Finally, the binding of aptamer with the target ligand is validated by surface plasmon resonance. (B) Competition assay was performed to determine whether P19 and P1 bind to the same epitope. The fluorescence intensity was quantified using confocal microscopy. *One-way ANOVA test; * P ≤ .05, ** P ≤ .01. (C) Ten percent of polyacrylamide gel electrophoresis was used to separate immobilized protein samples after pull-down with biotinylated P19, P1, and negative control RNAs. Coomassie-stained gels: M (marker), P19 (lane 1), P1 (lane 2), and NC (irrelevant RNA, lane 3). Arrow indicates target antigens. (D) MS/MS spectra of aptamer binding ligand. Peptide matching and MS/MS spectra of P19 (left) and P1 (right) affinity-purified peptides. Inset: amino acid sequence of the parent peptide showing b- and y-ion series coverage. Target epitopes are highlighted in yellow. (E) Biosensor analysis of the mortalin-aptamer interaction. Binding of mortalin to the P19 (left) and P1 (right) was shown. Biotinylated P19 or P1 was bound to a streptavidin-coupled carboxyl methyl dextran surface, and binding was measured using the SPR technique. The increased response unit was shown in a dose-dependent manner.

Article Snippet: Recombinant mortalin was purchased from Novus Biologicals (NBC1-18380).

Techniques: Clinical Proteomics, Membrane, In Vitro, Tandem Mass Spectroscopy, Binding Assay, SPR Assay, Competitive Binding Assay, Fluorescence, Confocal Microscopy, Polyacrylamide Gel Electrophoresis, Negative Control, Staining, Marker, Affinity Purification, Sequencing