Primary antibodies used for western blot were anti-choline kinase α, anti-Mfn2 (Abcam); anti-Drp1, anti-Opa1 (BD Biosciences); anti-Pak1, anti-Pak2, anti-phospho-Pak1(S144)/Pak2(S141), anti-pDrp1(S637), anti-Tom20 (Cell Signaling Technology); anti-choline kinase β, anti-Vdac1, anti-Mfn2 (Santa Cruz); and anti-Gapdh (Ambion). Secondary antibodies used were anti-mouse HRP IgG and anti-rabbit HRP (Cell Signaling Technology). For immunofluorescence, primary antibodies used were dystrophin and Ki67 (Abcam), Pax7 (DSHB), and laminin (Sigma). Alexafluor-488-conjugated wheat germ agglutinin and secondary antibodies (anti-mouse IgG1 Alexafluor-647 and anti-rabbit Alexafluor-488) were from Invitrogen. For references on use of selected antibodies, see [18 ] (Pak1, Pak2, pPak1/2, GAPDH); [27 (link)] (DRP1, pDRP1(S637), Opa1, Mfn1, Mfn2,); [28 (link)] (laminin); [29 (link)] (Ki67); and [30 (link)] (choline kinase α).
Anti mfn2
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
Anti-Mfn2 is a primary antibody that recognizes the Mitofusin-2 (Mfn2) protein. Mfn2 is a large GTPase that plays a critical role in mitochondrial fusion and dynamics. This antibody can be utilized in various research applications, including western blotting, immunoprecipitation, and immunofluorescence, to study the expression and localization of Mfn2 in different cell types and biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti mfn1, by Santa Cruz Biotechnology (6 mentions)
Anti drp1, by Santa Cruz Biotechnology (6 mentions)
Anti β actin, by Santa Cruz Biotechnology (5 mentions)
Anti opa1, by Santa Cruz Biotechnology (4 mentions)
Anti fis1, by Santa Cruz Biotechnology (3 mentions)
Anti phospho drp1 ser616, by Cell Signaling Technology (3 mentions)
Anti beclin 1, by Cell Signaling Technology (2 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (2 mentions)
Anti drp1, by Cell Signaling Technology (2 mentions)
Anti opa1, by Thermo Fisher Scientific (2 mentions)
Anti erk1 2, by Santa Cruz Biotechnology (2 mentions)
Anti p ampk, by Cell Signaling Technology (2 mentions)
Anti β tubulin, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 488 conjugated wheat germ agglutinin, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Thermo Fisher Scientific (1 mentions)
Laminin, by Merck Group (1 mentions)
Dystrophin, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti mfn2, by Abcam (1 mentions)
Anti tom20, by Cell Signaling Technology (1 mentions)
15 protocols using anti mfn2
1
Molecular Profiling of Mitochondrial Dynamics
2
Western Blot Analysis of Autophagy and Mitochondrial Dynamics
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Western blot was performed as previously described [17] (link). In brief, blocked membranes were incubated with anti-Beclin1, anti-LC3B (Cell Signaling Technology, Beverly, MA), anti-S100A7, anti-Mfn1, anti-Mfn2, anti-β-actin (Santa Cruz), or anti-DRP1 (BD Biosciences, San Jose, CA) at a dilution of 1∶1,000 or 1∶2,000. Immunoreactive bands were visualized using a chemiluminescent ECL detection kit (Pierce, Rockford, IL).
3
Western Blot Analysis of Mfn2 and ERK1/2
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Total protein was extracted in RIPA buffer (CxBio, Shanghai, China) supplemented with phenylmethanesulfonyl (PMSF, CxBio). The protein concentrations were measured using the BCA Protein Assay (Applygen, Beijing, China). The samples were separated on a 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane, which was blocked in 5% skim milk for 1 h and then incubated with primary antibodies at 4 °C overnight. After the washing steps, the membranes were incubated with horseradish peroxidase-labelled secondary antibodies for 1 h at room temperature. The bands were visualized using a chemiluminescence detection system, and the densitometric results were analysed with Image J software. EIF5 levels were used as internal controls for protein normalization.
Anti-Mfn2 and anti-EIF5 for western blotting were purchased from Santa Cruz Biotechnology (CA, USA), and anti-phospho-ERK1/2 and anti-total-ERK1/2 antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA).
Anti-Mfn2 and anti-EIF5 for western blotting were purchased from Santa Cruz Biotechnology (CA, USA), and anti-phospho-ERK1/2 and anti-total-ERK1/2 antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA).
4
Mitochondrial Dynamics Analysis in Cells
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Cells were lysed in RIPA lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1 mM phenylmethanesulfonyl fluoride; 1 mM ethylenediaminetetraacetic acid (EDTA); 1% Triton X-100; 1% sodium deoxycholate; and 0.1% SDS) with the addition of Protease Inhibitor Cocktail (Roche Diagnostics, Penzberg, Germany) and Phosphatase Inhibitor Cocktail I (Sigma, St. Louis, MO, USA). The antibodies used included anti-COX2 (Abcam, Cambridge, UK), anti-β-actin (Cell Signaling, Danvers, MA), anti-OPA1 (Santa Cruz Biotechnology), anti-MFN2 (Santa Cruz Biotechnology), anti-DRP1 (Santa Cruz Biotechnology), anti-FIS1 (Santa Cruz Biotechnology), and peroxidase-labeled anti-rabbit IgG (H + L) secondary antibody (Abcam, Cambridge, UK). The signals were developed using ECL plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) using X-ray films.
5
Mitochondrial Dynamics and Autophagy Analysis
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The hippocampus or cells were lysed in radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors and homogenized for 2 min. Protein concentrations were then measured using a Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, P0010). The loading volume was calculated according to a protein content per well of 40 μg; the experimental protocol was consistent with that of a previous study.27 After blocking with 5% Albumin Bovine V (Solarbio, A8020) in Tris‐buffered saline containing 0.1% Tween 20 for 1 h at room temperature, membranes were incubated overnight at 4°C with the following primary antibodies: anti‐Drp1, anti‐Fis1, anti‐OPA1, anti‐Mfn1, anti‐Mfn2 (Santa Cruz, CA, USA), anti‐p‐Drp1 (Ser637), anti‐p‐Drp1 (Ser616), anti‐cytochrome oxidase subunit IV (COXIV; Cell Signaling Technology), anti‐sequestosome 1, (p62; Cell Signaling Technology), anti‐microtubule‐associated protein 1 light chain 3β, (LC3B, Cell Signaling Technology and Abcam), and anti‐superoxide dismutase 2, (SOD2, Proteintech). Subsequent incubation was performed using goat anti‐rabbit/mouse secondary antibodies for 1 h at room temperature. The membranes were then imaged via enhanced chemiluminescence using an Odyssey infrared imaging system (LI‐COR Biosciences).
6
Quantitative Western Blot Analysis
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The western blotting procedures were fully described previously.
14 (link) The following primary antibodies were used: anti‐4‐HNE (Abcam, AB46545, 1:4000), anti‐Bax (Santa Cruz, Sc‐7480, 1:100), anti‐Bcl‐2 (Santa Cruz, Sc‐7382, 1:200), anti‐catalase (GeneTex, GTX110704, 1:1000), anti‐citrate synthase (Santa Cruz, Sc‐390693, 1:200), anti‐p‐DRP1 (Ser616, Cell Signaling, #34555, 1:500), anti‐DRP1 (Cell Signaling, #5391S, 1:500), anti‐Fis1 (Santa Cruz, Sc‐376447, 1:200), anti GPx1 (Thermo Fisher Scientific, PA5‐26323, 1:1000), anti‐MFN2 (Santa Cruz, Sc‐100560, 1:200), anti‐Mul1 (Abcam, AB209263, 1:1000), anti‐OPA1 (Thermo Fisher Scientific, MA5‐16149, 1:1000), anti‐Parkin (Abcam, AB77924, 1:2000), anti‐PINK1 (Thermo Fisher Scientific, PA1‐16604, 1:500), anti‐PGC‐1α1 (Millipore, AB3242, 1:1000), anti‐SOD2 (Genetex, GTX116093, 1:1000) and anti‐VDAC (Santa Cruz, Sc‐390996, 1:200). Then, anti‐rabbit (Cell Signaling, #7074S, 1:4000) or anti‐mouse (Cell Signaling, #7076S, 1:4000) secondary antibodies were used. The blots were revealed using a Pierce ECL kit (Thermo Fisher Scientific) or SupraSignal Femto kit (Thermo Fisher Scientific), and proteins were visualized by enhanced chemiluminescence (iBright 1500 Imaging System, Invitrogen) and quantified with ImageJ Software (version 1.8.0). Ponceau coloration was used as the loading control.
14 (link) The following primary antibodies were used: anti‐4‐HNE (Abcam, AB46545, 1:4000), anti‐Bax (Santa Cruz, Sc‐7480, 1:100), anti‐Bcl‐2 (Santa Cruz, Sc‐7382, 1:200), anti‐catalase (GeneTex, GTX110704, 1:1000), anti‐citrate synthase (Santa Cruz, Sc‐390693, 1:200), anti‐p‐DRP1 (Ser616, Cell Signaling, #34555, 1:500), anti‐DRP1 (Cell Signaling, #5391S, 1:500), anti‐Fis1 (Santa Cruz, Sc‐376447, 1:200), anti GPx1 (Thermo Fisher Scientific, PA5‐26323, 1:1000), anti‐MFN2 (Santa Cruz, Sc‐100560, 1:200), anti‐Mul1 (Abcam, AB209263, 1:1000), anti‐OPA1 (Thermo Fisher Scientific, MA5‐16149, 1:1000), anti‐Parkin (Abcam, AB77924, 1:2000), anti‐PINK1 (Thermo Fisher Scientific, PA1‐16604, 1:500), anti‐PGC‐1α1 (Millipore, AB3242, 1:1000), anti‐SOD2 (Genetex, GTX116093, 1:1000) and anti‐VDAC (Santa Cruz, Sc‐390996, 1:200). Then, anti‐rabbit (Cell Signaling, #7074S, 1:4000) or anti‐mouse (Cell Signaling, #7076S, 1:4000) secondary antibodies were used. The blots were revealed using a Pierce ECL kit (Thermo Fisher Scientific) or SupraSignal Femto kit (Thermo Fisher Scientific), and proteins were visualized by enhanced chemiluminescence (iBright 1500 Imaging System, Invitrogen) and quantified with ImageJ Software (version 1.8.0). Ponceau coloration was used as the loading control.
7
Purification and Immunoblotting of CRP
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Human C-reactive protein (CRP) was obtained from Millipore (Burlington, MA, USA). As sodium azide must be removed from commercial CRP before treatment, we filtered CRP with Tris buffer [10 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 2 mM Ca2+] until the remaining sodium azide was 0.0001%, using Amicon Ultra-0.5 filter (Millipore). Anti-DRP1, anti-MFN1, anti-MFN2, anti-Tom20, anti-ERK1/2, anti-phospho-ERK1/2 (Tyr 204), anti-PARK2, anti-β-actin, and anti-GAPDH antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-phospho-DRP1 (Ser616) and anti-OPA1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-VDAC1/Porin and anti-PINK1 antibodies were purchased from Abcam (Cambridge, UK) and Novus (Centennial, CO, USA), respectively. U0126 (an ERK 1/2 inhibitor) was obtained from Sigma-Aldrich (St. Louis, MO, USA).
8
Paraventricular nucleus protein analysis
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The paraventricular nucleus was lysed with IP lysis buffer (Beyotime, Nanjing, China) and then homogenized. The protein in the supernatant was quantified with a BCA assay kit (Pierce, Rockford, IL, USA), which was performed according to the manufacturer’s instructions. Equal amounts of protein were denatured and then analyzed with polyacrylamide gel electrophoresis. The separated proteins were transferred to a PVDF membrane. The membrane was blocked with plasma. Primary antibodies included anti-PGC-1α, anti-Mfn2, anti-Nrf2, anti-hemeoxygenase, anti-β-actin (Santa Cruz), anti-OxPhos Complex III or anti-Complex V (Invitrogen), anti-Beclin1, anti- AMPK, and anti-p-AMPK (Thr 172, Cell Signaling Technology, Beverly, MA). Primary antibodies were added and incubated with the membrane. After washing, the membrane was incubated with secondary antibody, and signals were observed by chemiluminescence. The result was photographed and analyzed with NIH Image J analysis software.
9
Mitochondrial Protein Expression Analysis
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The primary antibodies used were as follows: Anti-β-tubulin (sc-9104, Santa Cruz Biotechnology, Inc., 1:2,000), anti-Total OXPHOS Human WB Antibody Cocktail (ab110411, Abcam, Inc. 1:2,000), anti-COX IV (11967S, Cell Signaling, 1:1,000), anti-β-actin (sc-47778, Santa Cruz Biotechnology, Inc., 1:1,000), anti-Opa1 (PA1-16991, Thermo Fisher Scientific, 1:1,000), anti-Cyclophilin D (sc-376061, Santa Cruz Biotechnology, Inc., 1:1,000), anti-ANT (Santa Cruz biotechnology; 1:1,000), anti-Mfn1 (sc-50330, Santa Cruz Biotechnology, Inc. 1:1,000), anti-Mfn2 (sc-50331, Santa Cruz Biotechnology, Inc., 1:1,000), anti-phospho-DRP1 (Ser616) (4494, Cell Signaling, 1:1,000), anti-DRP1 (sc-271583, Santa Cruz Biotechnology, Inc., 1:1,000), anti-nitrotyrosine (141682, US Biological, Life Sciences, 1:500), and anti-4HNE (H6275-02, US Biological, Life Sciences, 1:1,000). The fluorescent dyes used were as follows: MitoTrackerTM Red CM-H2Xros (M7513, Thermo Fisher Scientific), MitoTrackerTM Green FM (M7514, Thermo Fisher Scientific), and VECTASHIELD Mounting Medium with DAPI (H1200, Vector Laboratories, Inc.).
10
Antibody-based Mechanistic Evaluation of Mitochondrial Dynamics
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The antibodies used in this study were as follows: anti-β-actin (sc-8432, Santa Cruz, CA, USA), anti-ERK1/2 (sc-135900, Santa Cruz, CA, USA), anti-p-ERK1/2 (sc-7383, Santa Cruz, CA, USA), anti-MFN1 (sc-50330, Santa Cruz, CA, USA), anti-RAGE (sc-365154, Santa Cruz, CA, USA and ab89911, Abcam, Cambridge, UK), anti-MFN2 (sc-100560, Santa Cruz, CA, USA), anti-OPA1(sc-393296, Santa Cruz, CA, USA), anti-phospho-Drp1Ser616 (#3455, Cell Signaling Technology, MA, USA), anti-phospho-Drp1 Ser637 (#6319, Cell Signaling Technology, MA, USA), anti-LC3 (#3868, Cell Signaling Technology, MA, USA), anti-HMGB1 (#3935, Cell Signaling Technology, MA, USA and #651401, Biolegend, San Diego, CA, USA), and anti-p62 (#39749, Cell Signaling Technology, MA, USA). All secondary antibodies (HRP-conjugated anti-rabbit, anti-mouse and anti-goat) were purchased from Santa Cruz Biotechnology. The lentivirus carrying shRNAs against RAGE, HMGB1, Drp1, MFN1 and OPA1 were obtained from the National Core Facility for Manipulation of Gene Function by RNAi, miRNA, miRNA sponges, and CRISPR/Genomic Research Center, Academia Sinica. The recombinant HMGB1 proteins, HMGB1 inhibitor quercetin were purchased from Sigma (St. Louis, MO, USA). RAGE inhibitor FPS-ZM1 was purchased from Merck (Temecula, CA, USA).
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