Sodium fluoride and orthovanadate, phenylmethylsulfonyl fluoride, aprotinin and leupeptin were purchased from Sigma (Saint-Louis, MO, USA). Anti-phospho-mTor, anti-mTor, anti-phospho-P70S6kinase (thr389), anti-P70S6kinase, anti-phospho-S6 Ribosomal Protein (ser235-236), anti-S6 Ribosomal Protein, anti-LC3-b, anti-phospho-DRP1 (ser616) and HRP conjugated anti-rabbit antibodies were from Cell Signaling Technology (Beverly, MA, USA). Anti-Hsp60, anti-Hsp90 and anti-DRP1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Smac antibody was purchased from RnD systems (Minneapolis, MN, USA). HRP-conjugated anti-mouse and anti-goat antibodies were from Dakopatts (Glostrup, Denmark).
Anti phospho drp1 ser616
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-DRP1 (Ser616) is a primary antibody that detects endogenous levels of DRP1 protein when phosphorylated at serine 616. It is used to analyze the phosphorylation state of DRP1 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti drp1, by Santa Cruz Biotechnology (3 mentions)
Anti opa1, by BD (3 mentions)
Anti dlp1, by BD (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti mfn1, by Santa Cruz Biotechnology (3 mentions)
Anti mfn2, by Santa Cruz Biotechnology (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Pure nitrocellulose membranes, by Pall Corporation (2 mentions)
Anti erk1 2, by Santa Cruz Biotechnology (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Hrp conjugated anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Anti s6 ribosomal protein, by Cell Signaling Technology (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Anti phospho mtor, by Cell Signaling Technology (1 mentions)
Anti hsp90, by Santa Cruz Biotechnology (1 mentions)
Anti phospho s6 ribosomal protein ser 235 236, by Cell Signaling Technology (1 mentions)
13 protocols using anti phospho drp1 ser616
1
Immunoblotting Analysis of Mitochondrial Proteins
2
Western Blot Analysis of Mitochondrial Proteins
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Proteins were extracted and blotted as previously described [40] (link). Primary antibody incubation was performed overnight at 4 °C (anti-UCP1, Calbiochem, #662045, dilution 1:750; and anti-β-tubulin, Sigma #T5201, dilution 1:2000; anti-DRP1, Cell Signaling #5391, dilution 1:1000; anti-phosphoDRP1(Ser616), Cell Signaling #4494, dilution 1:1000; anti-citrate synthase, Abcam #ab96600, dilution 1:10,000) and then detected with HRP-conjugated anti-rabbit or anti-mouse immunoglobulins (Promega, Charbonnières-les-Bains, France). Detection was performed using Chemiluminescent HRP Substrate (Millipore, Molsheim, France). OD band intensities were evaluated using PCBas Software.
For mitochondrial complex quantitation, equal amounts of cell proteins were separated using gradient SDS-PAGE (10–20%) and blotted onto nitrocellulose membranes. Saturated membranes were incubated overnight with a 1:1000 dilution of total OXPHOS human Western Blot Antibody Cocktail (#MS601, Mitosciences) followed by 60 min incubation with HRP-conjugated anti-mouse immunoglobulins. Chemiluminescence obtained after addition of Clarity Western ECL Substrate (BioRad, Marnes-la-Coquette, France) was detected using a ChemiDoc MP Imaging System (Bio-Rad) and quantified with Image Lab 5.0 software (Bio-Rad).
For mitochondrial complex quantitation, equal amounts of cell proteins were separated using gradient SDS-PAGE (10–20%) and blotted onto nitrocellulose membranes. Saturated membranes were incubated overnight with a 1:1000 dilution of total OXPHOS human Western Blot Antibody Cocktail (#MS601, Mitosciences) followed by 60 min incubation with HRP-conjugated anti-mouse immunoglobulins. Chemiluminescence obtained after addition of Clarity Western ECL Substrate (BioRad, Marnes-la-Coquette, France) was detected using a ChemiDoc MP Imaging System (Bio-Rad) and quantified with Image Lab 5.0 software (Bio-Rad).
3
Quantifying Mitochondrial Protein Expression
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15 to 70 μg of protein was electrophoresed on 10–15% vol/vol polyacrylamide SDS-PAGE gels. Proteins were electrophoretically transferred onto PVDF membranes. The membranes were subsequently blocked and, after blocking, were incubated at room temperature with the following antibodies: 1 : 500 anti-NOS1 (R-20) : sc-648, 1 : 500 anti-Mfn2 (H-68) : sc-50331, 1 : 2000 anti-actin (I-19) : sc-1616, and 1 : 2000 anti-VDAC1 (N-18) : sc-8828 were obtained from Santa Cruz, CA. 1 : 500 anti-OPA1 : 612607 and 1 : 1000 anti-DLP1 : 611113 were obtained from BD Biosciences. 1 : 1000 anti-phospho-DRP1 (Ser616) : #3455 was obtained from Cell Signaling. 1 : 1000 anti-nitrotyrosine, clone1A6 : #05-233 was obtained from EMD Millipore.
4
Western Blot Analysis of Mitochondrial Dynamics Proteins
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Primary hippocampal neurons were lysed in lysis buffer (10 mM Tris at pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 1% Triton X-100, 1% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Sigma-Aldrich) as previously described [40] (link). The extracted proteins were diluted into indicated amounts and separated on 8–12.5% SDS/PAGE and transferred onto polyvinylidene difluoride (PVDF) blotting membranes (BioRad). The membranes were blocked in 5% non-fat milk (Cell Signaling) dissolved in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 for 1 h at room temperature and probed with different antibodies: anti-DLP1 (1:3000, BD Biosciences), anti-phospho-Drp1 (Ser 616) (1:1000, Cell Signaling), anti-OPA1 (1:1000, BD Biosciences), anti-Mfn1 (1:1000, Novus Biologicals), anti-Mfn2 (1:1000, Sigma-Aldrich), anti-VDAC (1:1000, Cell Signaling), anti-cleaved-caspase-3 (Asp175) (1:1000, Cell Signaling), and anti-β-actin (1:10,000, Sigma-Aldrich). ECL (GE Healthcare) and Western Bright ECL (Advansta) were used to develop the film. Quantification analysis was performed on scanned films using Image J (NIH).
5
Western Blot Analysis of Oocyte Proteins
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Fifty denuded oocyte lysates in PRO-PREP protein lysis buffer (iNtRON, Daejeon, South Korea) were prepared. Oocyte lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to pure nitrocellulose membranes (Pall Life Sciences, NY, United States) after electrophoresis. The membranes were incubated with primary antibodies such as anti-phospho-DRP1-Ser616 (Cell Signaling), DRP1 (Santa Cruz Biotechnology), AC-TUBULIN (Cell Signaling), and β-actin (Santa Cruz Biotechnology). The membranes were incubated with a secondary horseradish peroxidase (HRP)-conjugated anti-rabbit/mouse IgG (Thermo Scientific, MA, United States). Binding antibodies were detected using the enhanced chemiluminescence (ECL) kit (Bio-Rad, CA, United States). The bands were visualized using Fusion Solo software (Vilber Lourmat, Collégien, France).
6
Western Blot Analysis of Phospho-Drp1 and AMPK
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Microglia in the primary culture were lysed using Laemmli sample buffer (0.5 Tris-HCl [pH 6.8] 12.5 mL, Glycerol 10 mL, 10% SDS 20 mL, 2-mercaptoethanol 50 μL and BPB) with 1% phosphatase inhibitor. Samples were subjected to SDS-PAGE and Western blotting. The primary antibodies used were anti-phospho-Drp1 (Ser616) (Cell Signaling Technology), anti-DLP1 (BD, 611112), anti-phospho-AMPKα (Thr172) (Cell Signaling Technology) and anti-AMPKα (Cell Signaling Technology) and all primary antibodies were diluted 1:1000. The second antibodies used were goat anti-rabbit IgG (H+L), HRP conjugate (Thermo, #31460) and HRP horse anti-mouse IgG antibody (Peroxidase) (Vector Laboratories, PI-2000) and all secondary antibodies were diluted 1:3000. We detected signals using chemiluminescence system.
7
Phospho-Drp1-Ser616 Immunoblotting in Embryos
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Cultured 40 embryos lysates in PRO-PREP protein lysis buffer (iNtRON, Daejeon, Korea) were prepared. Embryo lysates were separated by 10% SDS-PAGE. After electrophoresis, the separated proteins were transferred to pure nitrocellulose membranes (Pall Life sciences, NY, USA). After blocking, the membranes were incubated anti-phospho-Drp1-Ser616 (Cell signaling, MA, USA), Drp1 (Santa Cruz Biotechnology, CA, USA) and β-actin (Santa Cruz). The membranes were incubated with a secondary HRP-conjugated anti-rabbit/mouse IgG (Thermo, Scientific, MA, USA). Antibody binding was detected using the ECL kit (Advansta, CA, USA) according to the manufacturer’s instructions.
8
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).
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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