Journal: PLoS ONE

Article Title: The mitochondrial proteomic changes of rat hippocampus induced by 28-day simulated microgravity

doi: 10.1371/journal.pone.0265108

Figure Lengend Snippet: The protein changes of mitochondria of hippocampus after 28 days of tail suspension in rats.

Article Snippet: The primary antibodies used were aconitate hydratase (ACO2, 6571), dihydrolipoamide S-succinyltransferase (DLST, 11954), citrate synthase (CS, 14309), and VDAC (4661), and all of the antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: The mitochondrial proteomic changes of rat hippocampus induced by 28-day simulated microgravity

doi: 10.1371/journal.pone.0265108

Figure Lengend Snippet: (A) Protein levels of ACO2, DLST and CS were determined by immunoblotting. (B) The relative expressions of ACO2, DLST and CS were represented by the intensity ratio between the protein and the loading control (VDAC) in each lane. n = 4, error bars indicated standard deviations. *p<0.05; **p<0.01 compared with each control group.

Article Snippet: The primary antibodies used were aconitate hydratase (ACO2, 6571), dihydrolipoamide S-succinyltransferase (DLST, 11954), citrate synthase (CS, 14309), and VDAC (4661), and all of the antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: Western Blot

Journal: iScience

Article Title: Prevention and regression of megamitochondria and steatosis by blocking mitochondrial fusion in the liver

doi: 10.1016/j.isci.2022.103996

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-ACO2 , Cell Signaling Technology , Cat# 6571; RRID: AB_2890191.

Techniques: Recombinant, Western Blot, Protease Inhibitor, Electron Microscopy, Activity Assay, Staining, Software

Journal: Cells

Article Title: The Flagellin:Allergen Fusion Protein rFlaA:Betv1 Induces a MyD88− and MAPK-Dependent Activation of Glucose Metabolism in Macrophages

doi: 10.3390/cells10102614

Figure Lengend Snippet: Suggested molecular signaling events contributing to rFlaA:Betv1-mediated activation of BMDMs. Stimulation of BMDM with aggregated rFlaA:Betv1 results in both TLR5-dependent and -independent uptake, leading to the activation of both MyD88− and MAPK-mediated signaling cascades. rFlaA:Betv1-induced pro-inflammatory IL-6 and TNF-α secretion were shown to be MyD88− and MAPK-dependent, likely promoting the induction of IFN-γ-producing Th1 cells in BMDM:TC co-cultures. Western blot analysis confirmed rFlaA:Betv1 to trigger STAT3 activation, increasing the expression of HIF-1a, which is likely to be responsible for the observed IL-1β secretion. Metabolically, we found a MyD88-dependent phosphorylation of the mTOR target protein p70 S6 kinase, which together with HIF-1a likely mediated the observed shift towards enhanced glycolysis with increased levels of the glucose transporter Glut-1 and PFKFB3, and the induction of the Warburg effect while being paralleled by a decreased expression of mitochondrial proteins such as ACO2 and reduced oxygen consumption rates, suggesting a disrupted Krebs cycle in these cells. Finally, TLR5, MyD88, and MAPK activation were shown to be involved in the rFlaA:Betv1-induced production of the anti-inflammatory cytokine IL-10 that likely caused the observed suppression of rBet v 1-induced Th1 and Th2 responses in BMDM:TC co-cultures.

Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with the following primary antibodies from Cell Signaling Technologies overnight at 4 °C: phospho-MAPK Family Antibody Sampler Kit (#9910), NF-κB Pathway Sampler Kit (#9936), mTOR Substrates Antibody Sampler Kit (#9862), Glycolysis Antibody Sampler Kit (#8337), anti-Phospho-Stat3 (Ser727) antibody (#9134), anti-HIF-1a antibody (#14179), anti-ACO2 antibody (#6571), and loading control anti-histone H3 antibody (#12648, HRP Conjugate) or anti-GAPDH antibody (#8884, HRP Conjugate).

Techniques: Activation Assay, Western Blot, Expressing, Metabolic Labelling

Journal: Cancer research

Article Title: Transcriptional repression of SIRT3 potentiates mitochondrial aconitase activation to drive aggressive prostate cancer to the bone

doi: 10.1158/0008-5472.CAN-20-1708

Figure Lengend Snippet: (A) CRISPR-Cas9 mediated stable targeting of ACO2 gene in C4-2 cells using three independent sgRNAs were confirmed by immunoblotting Wild Type (WT) and Knockdown (ACO2-KD) pooled clones. Actin was used as a loading control.

Article Snippet: Immunohistochemistry Tumors were fixed and stained with primary antibodies ACO2 (Cell Signaling, 6571), Ki67 (Dako, M72K0), Firefly luciferase Antibody (Thermo, MA1-16880), and SIRT3 (Cell Signaling, 2627) followed by washing and incubated with secondary antibody (Thermo).

Techniques: CRISPR, Western Blot, Clone Assay

Journal: Cancer research

Article Title: Transcriptional repression of SIRT3 potentiates mitochondrial aconitase activation to drive aggressive prostate cancer to the bone

doi: 10.1158/0008-5472.CAN-20-1708

Figure Lengend Snippet: (A) C4-2-WT and C4-2-ACO2-KD cells (n=5, each group) were used to measure oxygen consumption rates (OCR) sequentially before and after the addition of oligomycin (2 μM), FCCP (1 μM), and antimycin A (1 μM) by XF24 Extracellular Flux Analyzer. Data are shown in mean ± S.D. Each time point is statistically significant. **** P<0.000001, two-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: Immunohistochemistry Tumors were fixed and stained with primary antibodies ACO2 (Cell Signaling, 6571), Ki67 (Dako, M72K0), Firefly luciferase Antibody (Thermo, MA1-16880), and SIRT3 (Cell Signaling, 2627) followed by washing and incubated with secondary antibody (Thermo).

Techniques:

Journal: Cancer research

Article Title: Transcriptional repression of SIRT3 potentiates mitochondrial aconitase activation to drive aggressive prostate cancer to the bone

doi: 10.1158/0008-5472.CAN-20-1708

Figure Lengend Snippet: (A) V5-tagged-ACO2 adenovirus were transduced to C4-2 cells fractionated into cytosolic (Cyto), mitochondrial (Mito), and nuclear (Nuc) compartments followed by immunoblotting with cytosolic, mitochondrial, and nuclear proteins β-tubulin, Tom20 and lamin A/C respectively.

Article Snippet: Immunohistochemistry Tumors were fixed and stained with primary antibodies ACO2 (Cell Signaling, 6571), Ki67 (Dako, M72K0), Firefly luciferase Antibody (Thermo, MA1-16880), and SIRT3 (Cell Signaling, 2627) followed by washing and incubated with secondary antibody (Thermo).

Techniques: Western Blot

Journal: Cancer research

Article Title: Transcriptional repression of SIRT3 potentiates mitochondrial aconitase activation to drive aggressive prostate cancer to the bone

doi: 10.1158/0008-5472.CAN-20-1708

Figure Lengend Snippet: (A) PC3 cells were transfected with myc-ACO2-WT or myc-ACO2-K258R for 48 hours (n=4, each group). Immunofluorescence staining was performed with anti-myc (green) while being counterstained with mitotracker (red) and DAPI (blue) followed by confocal microscopy. Scale bars, 10 μm.

Article Snippet: Immunohistochemistry Tumors were fixed and stained with primary antibodies ACO2 (Cell Signaling, 6571), Ki67 (Dako, M72K0), Firefly luciferase Antibody (Thermo, MA1-16880), and SIRT3 (Cell Signaling, 2627) followed by washing and incubated with secondary antibody (Thermo).

Techniques: Transfection, Immunofluorescence, Staining, Confocal Microscopy

Journal: Cancer research

Article Title: Transcriptional repression of SIRT3 potentiates mitochondrial aconitase activation to drive aggressive prostate cancer to the bone

doi: 10.1158/0008-5472.CAN-20-1708

Figure Lengend Snippet: (A) HEK293T cells were transduced with WT-SIRT3-Flag or HA-SIRT3-H248Y plasmid and immunoblotted for ACO2, HA, Flag, Actin, and Ac-K in input depicting WT-SIRT3-Flag or HA-SIRT3-H248Y expression. Transfected cell lysates were used for endogenous ACO2 and IgG (control) immunoprecipitation, followed by immunoblotting for ACO2 and Ac-K.

Article Snippet: Immunohistochemistry Tumors were fixed and stained with primary antibodies ACO2 (Cell Signaling, 6571), Ki67 (Dako, M72K0), Firefly luciferase Antibody (Thermo, MA1-16880), and SIRT3 (Cell Signaling, 2627) followed by washing and incubated with secondary antibody (Thermo).

Techniques: Transduction, Plasmid Preparation, Expressing, Transfection, Immunoprecipitation, Western Blot