rabbit polyclonal against vdac  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: Mapping the intracellular HMGB1 interactome and alterations induced by Toll-like receptor 4 activation

doi: 10.1016/j.jbc.2025.110866

Figure Lengend Snippet: The HMGB1 interactome in THP-1 cells changes after TLR4 activation. A , illustration of construct used to insert MycBioID2-HMGB1 into THP-1 cells. B , titration of doxycycline concentration to induce MycBioID2-HMGB1 expression. Lysates were analyzed by Western blotting using anti-Myc-HRP. GAPDH was used as loading control. C , Volcano plot of the HMGB1 interactome in resting (n = 3) and LPS-stimulated (n = 3) THP-1 cells. p -values were calculated by multiple paired two-way ANOVA corrected for multiple testing by FDR. D , the subcellular localization of proteins in the HMGB1 interactome. E , lysates from resting and LPS stressed THP-1 cells were fractioned into nuclear (N), cytosolic (C) and mitochondrial (M) fractions and analyzed by Western blotting using anti-HMGB1 and anti-BioID2. LaminB1, GAPDH and VDAC were used as loading controls (n = 3). All Western blots have been marked with molecular weights (kDa) on the right side.

Article Snippet: The following antibodies were used: 2040 (Mouse monoclonal-HRP against Myc-tag, Cell signaling technology), 3683 (Rabbit monoclonal-HRP against GAPDH, Cell signaling technology), 2G7 (Mouse monoclonal IgG2b against HMGB1 ( )), ab232733 (Mouse monoclonal IgG1 against BioID2, Abcam), 15068 (Rabbit monoclonal-HRP against LaminB1, Cell signaling technology), 4866 (Rabbit polyclonal against VDAC, Cell signaling technology), 7076 (affinity-purified horse anti-mouse IgG-HRP conjugate, Cell signaling technology) and 170-6515 (Goat anti-Rabbit IgG (H + L)-HRP-conjugate, Biorad).

Techniques: Activation Assay, Construct, Titration, Concentration Assay, Expressing, Western Blot, Control

Journal: iScience

Article Title: Protective roles of MITOL against myocardial senescence and ischemic injury partly via Drp1 regulation

doi: 10.1016/j.isci.2022.104582

Figure Lengend Snippet: MITOL knockout induces the Drp1-dependent fragmentation of mitochondria, followed by the vulnerability and senescence in MEFs (A) Accumulation of Drp1 in MITOL knockout (MITOL-KO) MEFs. Cre-ERT2-expressing WT MITOL (WT) or MITOL flox/flox MEFs were treated with 0.8 μM 4-hydroxytamoxifen (4-OHT) for one day. Four days after 4-OHT treatment was defined as 4d, and more than one month after 4-OHT treatment was defined as 1 M. Knockout of MITOL was confirmed and Drp1 expression was detected by immunoblot (IB) analysis with anti-MITOL and anti-Drp1 antibodies, respectively. Error bars represent ±SEM (n = 4). Analysis was performed with one-way ANOVA followed by Bonferroni post hoc analysis. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (B) MITOL ubiquitinates Drp1. MITOL F/F MEFs (control) or MITOL-KO MEFs were co-transfected with/without indicated expression vectors for FLAG-tagged Drp1, HA-tagged ubiquitin and non-tagged MITOL for 24 h and lysates were immunoprecipitated (IP) with anti-FLAG antibody, followed by immunoblotting with anti-HA antibody or anti-FLAG antibody. Cells were treated with MG132 (10 μM) for 10 h before harvest. Whole lysates were immunoblotted with anti-MITOL and anti-tubulin antibodies. (C) Accumulation of Drp1 in mitochondrial fraction. IB assay was performed on lysates of whole (WCL), cytosolic (Cyt/Micro), and mitochondrial (mito) fractions isolated from MITOL F/F MEFs (control) or MITOL-KO (KO) MEFs with Drp1 antibody. Anti-VDAC and anti-tubulin antibodies were used as a mitochondrial marker and a cytosolic marker, respectively. Mitochondrial Drp1 was normalized by the intensity of VDAC. Error bars represent ±SEM (n = 3). ∗∗∗p < 0.001 (Student’s t -test). (D) Mdivi-1 inhibits Drp1 accumulation in MITOL-KO mitochondria. MITOL-KO MEFs were treated with or without 5 μM Mdivi-1 for 12 h and lysates of whole and mitochondrial fractions were immunoblotted with anti-Drp1 antibody. Anti-Tom20 and anti-tubulin antibodies were used as a mitochondrial marker and a cytosolic marker, respectively. Mitochondrial Drp1 was normalized by the intensity of Tom20. Error bars represent ±SEM (n = 3). ∗∗∗p < 0.001 (Student’s t-test). (E and F) Mdivi-1 attenuates mitochondrial fragmentation in MITOL-KO MEFs. MITOL F/F MEFs (control), MITOL-KO MEFs (MITOL KO), and MITOL-KO MEFs treated with 5 μM Mdivi-1 for 12 h (MITOL KO + Mdivi-1) were stained with MitoTracker Green and mitochondrial morphologies were compared. Bar, 10 μm (E). Percentages of cells showing each mitochondrial morphology were calculated from 100 cells of each MEFs shown in E. Mean ± SEM (n = 3). Analysis was performed with two-way ANOVA followed by Bonferroni post hoc analysis. ∗∗∗∗p < 0.0001 (F). (G) Mdivi-1 attenuates mitochondrial ROS production in MITOL-KO MEFs. MITOL flox/flox MEFs (control) or MITOL-KO (KO) MEFs treated with or without 5 μM Mdivi-1 for 12 h were stained with MitoSOX and mitochondria-derived superoxide generation was measured by flow cytometric analysis. Bar graphs show relative levels of mean fluorescence intensity of MitoSOX compared with that of MITOL flox/flox MEFs (control). Mean ± SEM (n = 3). Analysis was performed with one-way ANOVA followed by Bonferroni post hoc analysis. ∗p < 0.05, ∗∗p < 0.01. (H and I) Accumulation of senescent cells in MITOL knockout MEFs was restored by Mdivi-1 treatment. Cytochemical staining of SA-β-gal activity in MITOL flox/flox MEFs (control), or MITOL-KO MEFs treated with or without 5 μM Mdivi-1 for six hours. Bar, 50 μm (H). The bar graph shows the percentages of SA-β-gal positive cells (100 cells/experiment, n = 3). Mean ± SEM. Analysis was performed with one-way ANOVA followed by Bonferroni post hoc analysis. ∗∗∗∗p < 0.0001 (I). (J) MITOL-KO MEFs promote age-related hypertrophy. The areas of MITOL flox/flox MEFs (control) and MITOL-KO MEFs were measured. Percentages of cells showing each cell size were calculated from 100 cells of each MEFs. Mean ± SEM (n = 3). ∗p < 0.05. (K) Mitochondrial fragmentation correlates with age-related hypertrophy in MITOL-KO MEFs. MITOL-KO MEFs were stained by anti-Tom20 and anti-Actin antibodies. Percentages of cells showing each mitochondrial morphology were calculated from 100 cells of each MEFs. Mean ± SEM (n = 4). ∗p < 0.05.

Article Snippet: The rabbit polyclonal antibodies against VDAC (#4866), Caspase-3 (#9662) and cleaved Caspase3 (#9661) were from Cell Signaling.

Techniques: Knock-Out, Expressing, Western Blot, Control, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Isolation, Marker, Staining, Derivative Assay, Fluorescence, Activity Assay

Journal: iScience

Article Title: Protective roles of MITOL against myocardial senescence and ischemic injury partly via Drp1 regulation

doi: 10.1016/j.isci.2022.104582

Figure Lengend Snippet: Mitochondrial morphological change by accumulated Drp1 and dysfunction in heart-specific MITOL-KO mice (A and B) Heart-specific MITOL KO enhances Drp1 accumulation in the heart. Samples were collected from the hearts of MITOL flox/flox (Control) and MITOL flox/flox ;αMHC-Cre Mer (MITOL cKO) mice treated with tamoxifen for the indicated periods, followed by immunoblotting with indicated antibodies. The relative protein levels of Drp1 were quantified by densitometry. Data are standardized to tubulin levels and are expressed relative to control mice. Mean ±SEM (n = 3). Analysis was performed with two-way ANOVA followed by Bonferroni post hoc analysis. ∗∗∗p < 0.001, ∗∗p < 0.01. (C and D) Abnormal morphologies of mitochondria in MITOL-KO mouse cardiomyocytes. Representative electron microscopic images of mitochondria from the hearts of MITOL flox/flox (Control) and MITOL flox/flox ;αMHC-Cre Mer (MITOL cKO) mice treated with tamoxifen for the indicated periods. 27,600-fold magnification (C). Mitochondrial size was represented as median surface area, and frequency distributions of mitochondrial surface were calculated from mitochondria imaged by TEM (D). Kruskal-Wallis test, ∗∗∗∗p < 0.0001. Data are median values. Bar, 500 nm. (E) Decreased size of mitochondria in MITOL-KO mouse cardiomyocytes. Cardiac mitochondrial areas of MITOL flox/flox (Control), and MITOL flox/flox ;αMHC-Cre Mer (MITOL cKO) mice treated with tamoxifen for the indicated periods were measured. Mitochondrial fractions isolated from cardiomyocytes of MITOL flox/flox and MITOL flox/flox ;αMHC-Cre Mer mice treated with tamoxifen for the indicated periods were stained with MitoTracker, followed by flow cytometric analysis. Bar graphs show the relative levels of mean fluorescence intensity of forward-scatter (FSC) and MitoTracker to MITOL flox/flox mice. Mean ±SEM (n = 3). ∗∗∗p < 0.001. (F) Mitochondrial ROS was upregulated in MITOL-KO mouse cardiomyocytes. Cardiac mitochondrial fractions of MITOL flox/flox (Control), and MITOL flox/flox ;αMHC-Cre Mer (MITOL cKO) mice treated with tamoxifen for the indicated periods were stained with MitoSOX and mitochondrial-derived superoxide generation was measured by flow cytometric analysis. Bar graph shows the relative levels of mean fluorescence intensity of MitoSOX to MITOL flox/flox mice. Mean ± SEM (n = 3). Analysis was performed with two-way ANOVA followed by Bonferroni post hoc analysis. ∗∗p < 0.01. (G and H) MITOL knockout induces mitochondrial oxidative damage in cardiomyocytes. Protein carbonyl contents of cardiac mitochondrial extracts were determined by protein carbonyls western blot detection kit (G). The levels of carbonylated proteins were quantified by densitometry. Data are standardized to VDAC levels and are expressed relative to MITOL flox/flox (Ctrl) mice prior to tamoxifen treatment. Mean ±SEM (n = 3). Analysis was performed with two-way ANOVA followed by Bonferroni post hoc analysis. ∗p < 0.05, ∗∗∗p < 0.001. (H). (I) Reduced oxygen consumption rate of mitochondria isolated from MITOL-KO mouse cardiomyocytes. The respiratory control ratio (RCR) represents the mitochondrial coupling state. Mean ±SEM (n = 3). ∗p < 0.05, Student’s t-test. (J) Reduced ATP content in the MITOL-KO heart. ATP content was measured by luciferase assay. Mean ±SEM (n = 4). ∗∗p < 0.01, Student’s t -test.

Article Snippet: The rabbit polyclonal antibodies against VDAC (#4866), Caspase-3 (#9662) and cleaved Caspase3 (#9661) were from Cell Signaling.

Techniques: Control, Western Blot, Isolation, Staining, Fluorescence, Derivative Assay, Knock-Out, Luciferase

Journal: iScience

Article Title: Protective roles of MITOL against myocardial senescence and ischemic injury partly via Drp1 regulation

doi: 10.1016/j.isci.2022.104582

Figure Lengend Snippet: Downregulation of MITOL in infarct cardiomyocytes and transduction of AAV-MITOL ameliorates cardiac fibrosis under myocardial infarction (A and B) Downregulation of MITOL in infarct rat cardiomyocytes. MI was induced by ligation of the left coronary artery in rats. After two weeks of MI, IB assay of MITOL and Drp1 was performed on lysates of whole and mitochondrial fractions isolated from rat hearts. Anti-VDAC and anti-tubulin antibodies were used as a mitochondrial marker and a cytosolic marker, respectively. Data are standardized to VDAC levels and are expressed relative to sham operated rats. Mean ±SEM (n = 4). ∗∗∗p < 0.001, Student’s t -test. (C) MITOL decreased in infarct cardiomyocytes of humans. Lysates from total heart cells of normal and infarct left ventricle were immunoblotted with anti-MITOL, anti-Drp1 and anti-tubulin antibodies. Age: 67–70. (D) Study design for the transduction of MITOL in MI-induced cardiomyocytes. Two days after the induction of MI, rats were introduced with AAV vectors (GFP: AAV-GFP and GFP-P2A-MITOL: AAV-MITOL) packaged in AAV-9 capsids via intrathoracic injection. Two weeks after virus injection, hearts were dissected and the expression of GFP and MITOL was assessed by immunoblotting. (E and F) MITOL was upregulated in cardiomyocytes of rats transduced with AAV-MITOL. IB assay of MITOL, GFP and tubulin was performed on lysates of total rat heart cells (E). The relative protein levels of MITOL and GFP were quantified by densitometry (F). Data are standardized to tubulin levels and are expressed relative to AAV-GFP injected rats. Mean ± SEM (n = 3). ∗∗p < 0.01. (G) MI-induced MITOL downregulation was rescued by AAV-MITOL. After MI or sham operation, rats were injected with indicated viruses (as described in D). Two weeks after injection of viruses, IB assay of MITOL, Drp1 and VDAC were performed on lysates of whole and mitochondrial fractions of rat cardiomyocytes. Data are standardized to VDAC levels and are expressed relative to AAV-GFP injected rats. (H and I) Mitochondria damage induced by MI was rescued by AAV-MITOL. Representative electron microscopic images of mitochondria from the hearts of sham-operated, MI treated, and MI +AAV-MITOL-treated mice. 27,600-fold magnification (H). Bar, 500 nm. The distribution of mitochondrial size is shown (I). (J and K) Myocardial fibrosis was attenuated in MI-treated mice transduced with AAV-MITOL. Representative photographs show Masson’s trichrome staining for collagen(J). Mean ±SEM (n = 4) (K). ∗∗p < 0.01. (L) Cardiac malfunction was restored in MI-treated mice transduced with AAV-MITOL. Echocardiographic analysis of left ventricular dimensions and cardiac function in mice. FS, fractional shortening of left ventricular diameter. Mean ±SEM (n = 3). Analysis was performed with one-way ANOVA followed by Bonferroni post hoc analysis. ∗p < 0.05. (M) Gene expression of markers of cardiac function and hypertrophy. Relative mRNA abundance of Tumor Necrosis Factor α (TNF-α), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP; B), α-myosin heavy chain (α-MHC), β-MHC, collagen-1 (Col-1), MHC-β, α-smooth muscle actin (α-SMA), transforming growth factor β (TGF-β), and fibronectin (FN) in the myocardium of control mice and MITOL cKO mice treated with AAV-GFP or with AAV-MITOL tested by real-time qPCR. Data are presented as mean ± SEM. ∗p < 0.05 vs. control. Analysis was performed with one-way ANOVA followed by Bonferroni post hoc analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (N) Gene expression of markers of cardiac aging. Relative mRNA abundance of P21 and Interleukin-1 (IL-1b) in the myocardium of control mice and MITOL cKO mice treated with AAV-GFP or with AAV-MITOL tested by real-time qPCR. Data are presented as mean ± SEM. ∗p < 0.05 vs. control. Analysis was performed with one-way ANOVA followed by Bonferroni post hoc analysis. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The rabbit polyclonal antibodies against VDAC (#4866), Caspase-3 (#9662) and cleaved Caspase3 (#9661) were from Cell Signaling.

Techniques: Transduction, Ligation, Isolation, Marker, Injection, Virus, Expressing, Western Blot, Staining, Gene Expression, Control

Journal: iScience

Article Title: Protective roles of MITOL against myocardial senescence and ischemic injury partly via Drp1 regulation

doi: 10.1016/j.isci.2022.104582

Figure Lengend Snippet:

Article Snippet: The rabbit polyclonal antibodies against VDAC (#4866), Caspase-3 (#9662) and cleaved Caspase3 (#9661) were from Cell Signaling.

Techniques: Marker, Recombinant, Reverse Transcription, Staining, Luciferase, In Situ, Software

Journal: The Biochemical journal

Article Title: The effect of chronic alcohol consumption on mitochondrial calcium handling in hepatocytes

doi: 10.1042/BCJ20160255

Figure Lengend Snippet: (A and B) Representative confocal images showing the lipid droplets stained with 1,6-diphenyl-1,3,5-hexatriene in hepatocytes isolated from control (A) or alcohol-fed rats (B) and maintained in primary culture for 1 h. (C) Biochemical determination of triglyceride levels in hepatocytes from control and alcohol-fed rats. Data are the means ± SEM, n = 4 pairs; *P< 0.05. (D and E) Immunoblot bands of mitochondrial respiratory chain polypeptides: (D) subunit NDUFA9 of complex I and (E) subunit NDUFB8 of complex I and subunit MTCO1 of complex IV. (F) Total ATP levels determined in hepatocytes from control and alcohol-fed rats incubated in the presence of 5 mM glutamate and 1 mM pyruvate. Data are the means ± SEM, n = 5 pairs; *P < 0.05. Immunoblot bands of VDAC (G) or Hsp10 and Hsp60 (H) in hepatocytes isolated from control and alcohol-fed rats. The numbers following the control and alcohol labels refer to a specific pairs of animals, i.e. the whole cell lysates used in E and H were from the same animals. Densitometric analysis of immunoblot bands are shown in Table 2.

Article Snippet: Rabbit polyclonal antibody against MCU/CCDC109A was obtained from Sigma, rabbit polyclonal antibody against voltage-dependent anion channel (VDAC) was from Cell Signaling, mouse monoclonal antibody against NDUFA9 was from Molecular Probes, mouse monoclonal antibodies against CypD/CypF and mitochondrial respiratory chain polypeptides were from Abcam and rabbit monoclonal antibodies against Hsp10 and Hsp60 were from Epitomics.

Techniques: Staining, Isolation, Control, Western Blot, Incubation

Journal: The Biochemical journal

Article Title: The effect of chronic alcohol consumption on mitochondrial calcium handling in hepatocytes

doi: 10.1042/BCJ20160255

Figure Lengend Snippet: Fold changes in the levels of mitochondrial proteins after chronic alcohol feeding Protein levels of the indicated mitochondrial proteins were determined in whole cell lysates prepared from hepatocytes isolated from control and alcohol-fed animals. Immunoblot bands were quantified by densitometry analysis using Image J (NIH). Representative examples of immunoblots are shown in . Data shown are the means ± SEM from 3 to 6 pairs of alcohol-fed rats and their pair-fed littermate controls. Proteins levels are normalized to TUBA or ACTB as indicated.

Article Snippet: Rabbit polyclonal antibody against MCU/CCDC109A was obtained from Sigma, rabbit polyclonal antibody against voltage-dependent anion channel (VDAC) was from Cell Signaling, mouse monoclonal antibody against NDUFA9 was from Molecular Probes, mouse monoclonal antibodies against CypD/CypF and mitochondrial respiratory chain polypeptides were from Abcam and rabbit monoclonal antibodies against Hsp10 and Hsp60 were from Epitomics.

Techniques: Isolation, Control, Western Blot