The p-mito-mRFP-RFP and pFLAG-CMV-HCV core DNA plasmids used in this study were described previously (8 (link), 15 (link), 16 (link)). The primary antibodies used in this study include the following: rabbit monoclonal anti-Drp1 (Cell Signaling), rabbit monoclonal anti-phospho-Drp1 (Ser616) (Cell Signaling), mouse monoclonal anti-HCV core (Thermo Scientific), rabbit polyclonal anti-β-actin (Cell Signaling), mouse monoclonal anti-TOM20 (BD Biosciences), human monoclonal anti-HCV E2 (17 (link)), mouse monoclonal anti-HCV NS3 (Abcam), mouse monoclonal anti-mitofusin 2 (Mfn2) (Abcam), and rabbit polyclonal anti-VDAC1 (Cell Signaling). The secondary antibodies used for Western blot analysis were HRP-conjugated anti-mouse IgG and HRP-conjugated anti-rabbit IgG (both from Promega). The secondary antibodies for immunofluorescence were Alexa Fluor 350, 488, 594, or 647 donkey anti-mouse, rabbit, or goat IgG and Alexa Fluor 555 goat anti-human IgG (all from Molecular Probes). The chemical reagents used in this study were carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Sigma), Sofosbuvir (Selleckchemm) and ginsenoside compounds (Sigma).
Anti vdac1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-VDAC1 is a primary antibody product that detects the VDAC1 (Voltage-Dependent Anion-Selective Channel Protein 1) protein. VDAC1 is a mitochondrial outer membrane protein that functions as a channel for the transport of small molecules and ions across the mitochondrial membrane. The Anti-VDAC1 antibody can be used to identify and study the VDAC1 protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti cleaved parp, by Cell Signaling Technology (2 mentions)
Anti hk2, by Cell Signaling Technology (2 mentions)
Anti bax, by Cell Signaling Technology (2 mentions)
Anti cytochrome c, by Cell Signaling Technology (2 mentions)
Anti oxphos, by Abcam (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
Anti cytochrome c, by Santa Cruz Biotechnology (2 mentions)
Anti parp, by Cell Signaling Technology (2 mentions)
Carbonyl cyanide m chlorophenylhydrazone cccp, by Merck Group (1 mentions)
Mouse monoclonal anti hcv core, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti tom20, by BD (1 mentions)
Hrp conjugated anti mouse igg, by Promega (1 mentions)
Hrp conjugated anti rabbit igg, by Promega (1 mentions)
Smmc 7721, by National Collection of Authenticated Cell Cultures (1 mentions)
Bel 7402, by National Collection of Authenticated Cell Cultures (1 mentions)
Hcclm3, by National Collection of Authenticated Cell Cultures (1 mentions)
Anti mouse igg hrp, by Cell Signaling Technology (1 mentions)
12 protocols using anti vdac1
1
Investigating Mitochondrial Dynamics and HCV Interactions
2
Chrysin Impacts Liver Cancer Cells
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Chrysin and other chemical reagents such as Tris, NaCl, SDS and DMSO were purchased from Sigma-Aldrich (St. Louis, MO). The normal human hepatic cell LO2 and HepG2, Hep3B, Huh-7, HCC-LM3, Bel-7402 and SMMC-7721 were obtained from the Cell Bank of Chinese Academy of Sciences and cultured with Dulbecco’s Modified Eagle Medium containing 10% FBS and 1% antibiotics in a 37 °C incubator with 5% CO2. Anti-HK2, anti-VDAC1, anti-cleaved-caspase3, anti-cleaved-PARP, anti-cytochrome C, anti-Bax, anti-Bak, anti-Bcl-2, anti-Bcl-xL, anti-rabbit IgG-HRP and anti-mouse IgG-HRP antibodies were products of Cell Signaling Technology, Inc. (Danvers, MA). Anti-α-Tubulin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin (A5316) was product of Sigma (St. Louis, MO, USA). HK2 (ORF004940) construct was purchased from Applied Biological Materials (ABM) Inc. (Richmond, BC, Canada). Lipofectamin 2000 was purchased from Invitrogen (Carlsbad, CA).
3
Indirubin Screening and Compound Characterization
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The chemical compounds derived from CMAP screening were purchased from Chemical Biology Technology Platform of Chinese Academy of Sciences (shanghai, china) and applied in a dose of 10 μM (final concentration) for each drug within cell experiments. Indirubin used in animal treatment was purchased from MedChemExpress (Catalog No.: HY-N117; CAS No.: 479–41-4; Purity: >98%, Monmouth Junction, NJ). Indirubin was first dissolved in dimethyl sulfoxide (DMSO) and stored in the dark at − 20 °C. During our experiments, Indirubin was diluted into corresponding concentration with the Corn oil (Sigma-Aldrich). All the other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise specified. The primary antibodies included anti-UCP1 (Abcam, ab10983 and ab23841), anti-PGC1α (Santa Cruze Biotechnology, sc-13,067), anti-OXPHOS (Abcam, ab110413), anti- phospho-(Ser/Thr) PKA Substrate (Cell Signaling Technology, #9621), anti-phospho-CREB (Cell Signaling Technology, #4276S, San Antonio, TX, USA), anti-VDAC1 (Cell Signaling Technology, #4661), anti-β-Tubulin (Cell Signaling Technology, #2146).
4
Investigating Autophagy and Oxidative Stress
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ZnO NPs, dansylcadaverine (MDC), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and the Mitochondrial Membrane Potential Kit were purchased from Sigma Chemical (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium and α-MEM culture media, the antibiotics–antimycotic solution, and fetal bovine serum were purchased from GIBCO (Grand Island, NY, USA). Anti-LC3B, anti-VDAC1, anti-p53, anti-caspase 9, and anti-GAPDH antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-PINK1 (ab23707) and anti-parkin (ab77924) were purchased from Abcam (Cambridge, MA, USA). MitoTracker Red CMXRos and Lipofectamine 2000 (11668-027) were purchased from Invitrogen (Carlsbad, CA, USA). GFP-LC3 was supplied by Prof Tanfeng from Wenzhou Medical University.
5
Cardiomyocyte Ndufs4 expression analysis
6
Protein Extraction and Western Blotting
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Cells were washed in cold Dulbecco's phosphate-buffered saline (DPBS) and lysed in Modified Oncogene Science lysis buffer (MOSLB) (50 mM NaPyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 μM Na3VO4, 10 mM HEPES, and 0.1%Triton X-100). Protein concentration was measured by either Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA; #500-0006) or BCA protein assay (Thermo Scientific, Rockford, IL, USA; 23227). SDS-PAGE was carried out with 20–40 μg of protein loading. Western blotting was performed according to standard protocol as previously described. The antibodies used in this study were anti-SQSTM1 (PROGEN Biotechnik GmbH, Heidelberg, Germany; GP62-C), anti-cytochrome c (Santa Cruz Biotechnology, H-104, sc-7159), anti-TOMM20 (Santa Cruz Biotechnology, FL-145, sc-11415), anti-NBR1 (Santa Cruz Biotechnology, sc-130380), anti-ACTB/β-actin (Sigma, Oakville, ON, Canada; A5316), anti-HA (Roche, Mississauga, ON, Canada; 11867423001), anti-PARK2 (Cell Signaling, Danvers, MA, USA; Prk8, #4211), anti-VDAC1 (Cell Signaling; #4866), and anti-MAVS (Cell Signaling, #8348), and anti-PINK1 (GeneTex, N3C3, Irvine, CA, USA; GTX107851). Protein levels were quantitated by densitometric analysis using NIH ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/ ) and normalized to those of ACTB.
7
Dioscin and MG132 Inhibit Colorectal Cancer
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Dioscin (≥95%) and MG132 were products of Sigma-Aldrich (St. Louis, MO). Normal colon epithelial cells FHC and CCD-18Co, and human colorectal cancer cells, including HCT116, HT29, DLD1, and SW620, were from the American Type Culture Collection (ATCC). All cells were cultured by following the standard protocols. Anti-HK2, anti-HK1, anti-VDAC1, anti-FBW7, anti-c-Myc, anti-cleaved-PARP, anti- cytochrome C, anti-cleaved-caspase-3, anti-Bax, anti-α-Tubulin, anti-β-actin, anti-mouse/rabbit secondary antibodies were from Cell Signaling Technology, Inc. (Danvers, MA). Anti-Ki67 and anti-c-Myc antibodies were purchased from Abcam (Cambridge, UK). Lipofectamine was from Invitrogen (Carlsbad, CA). HK2 overexpression plasmid was purchased from Addgene (Cat.: 23854). Control siRNA (Cat.: sc-37007) and FBW7 (Cat.: sc-37547) siRNAs were product of Santa Cruz.
8
Protein Extraction and Western Blot Analysis
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Total protein was extracted with RIPA buffer containing phenylmethylsulfonyl fluoride (PMSF) and Halt Protease and Phosphatase Inhibitor Cocktail. Membrane protein was extracted by membrane and cytosol protein extraction kit (Beyotime Biotechnology, China). The concentration of proteins was tested using the bicinchoninic acid (BCA) protein assay. Protein samples (30 μg) were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Bio-Rad, Richmond, CA, USA). Membranes were blocked in 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at room temperature. Then, membranes were incubated with primary antibody at 4 °C overnight. Anti-MTPα, anti-IRS1/P-IRS1, anti-Glut4, anti-β-actin, anti-GAPDH, anti-SIRT1, anti-Ace, anti-Ub and anti- Na+-ATPase α-1 were from Abcam. Anti-Akt/P-Akt, anti-VDAC1, anti-H2B and secondary antibody were from Cell Signaling Technology. Probed membranes were washed several times with TBST, and then incubated with horseradish peroxidase conjugated secondary antibodies at room temperature for 1 h. Bound antibody was detected with enhanced chemiluminescence (Millipore, Billerica, MA, USA). Protein expression was quantified using Image J software (NIH, USA). Total protein expression was normalized with respect to β-actin/GAPDH expression and membrane protein was normalized with respect to Na+-ATPase α-1 expression.
9
Western Blot Antibody Analysis
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Western blot was performed as described in our previous study 27 . Antibodies employed in this study were: anti-TRIP-Br1 (Enzo Life Sciences, ALX-804-645), anti-PARP (Cell Signaling Technology, #9542S), anti-cytochrome c (Santa Cruz Biotechnology, sc-7159), anti-CypA (Enzo Life Sciences, BML-SA296), anti-LC3 (Cell Signaling Technology, #2775S), anti-VDAC1 (Cell Signaling Technology, #4661), anti-TOMM20 (BD biosciences, 612278), anti-TIMM23 (BD biosciences, 611222), anti-HSP60 (Santa Cruz Biotechnology, sc-139661), anti-HSP70/GRP75 (Santa Cruz Biotechnology, sc-13967), anti-cathepsin B (Bioworld, BS3536), anti-cathepsin D (Bioworld, BS90201), anti-LAMP1 (Santa Cruz Biotechnology, sc-20011), anti-rabbit (Cell Signaling Technology, #7074S), and anti-mouse (Santa Cruz Biotechnology, sc-516102). Antibodies against γ-tubulin (Santa Cruz Biotechnology, sc-7396) and β-actin (Santa Cruz Biotechnology, sc-47778) were used to measure levels of γ-tubulin and β-actin as loading controls. Results of western blot analysis were semi-quantified using ImageJ software (ver. 1.51u; National Institutes of Health, USA). The relative intensity was compared to γ-tubulin or β-actin level and presented as bar graphs.
10
Western Blot Analysis of Cellular Proteins
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H9C2, NRVMs, or heart homogenates were lysed in RIPA buffer (50 mM Tris pH 7.2, 500 mM NaCl, 1% Triton X-100, 1 mM EDTA, protease inhibitors (Roche 11873580001), phosphatase inhibitors (Sigma-Aldrich, P0044 and P5726). Equal protein amounts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10-15% gels depending upon protein's molecular weights. After electrophoresis, proteins were transferred to nitrocellulose membranes and incubated with the following antibodies: anti-VDAC1 (#4866), anti-H3 (#9715), anti-HA (#3724) anti-GAPDH (#5174), anti-LC3B (#2775), anti-cleaved caspase 3 (#9661) and anti-p-p53 (#12571) from Cell Signaling Technology, anti-p62 (#H00008878-M01) from Abnova, anti-CTSD (#ab75852), anti-4-HNE (#ab46545) and anti-Oxphos (#ab110413) from Abcam. Proteins were detected by chemiluminescence with a Bio-Rad ChemiDoc XRS+ camera. Relative densities were quantified using the ImageLab 5.2.1 software (Bio-Rad). All data were normalized to internal controls.
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