Real-time PCR analysis was performed as previously described with primers and probe sets from Applied Biosystems31 (link). Immunoblots were performed and quantified as described previously12 (link), using the following antibodies: NF-κB2 (#4882), RelB (#4954), p-Mst1/2 (#3681), p-Yap (Ser127) (D9W2I), Lats1 (C66B5), Mst1 (D8B9Q), Mst2 (#3952), Actin (8H10D10), p-DRP1 (Ser637) (#4867), p-S6 (2F9), c-Myc (#9402s) (all from Cell Signaling Technology), OPA1 (NB110-55290SS, Novus Biologicals) and MitoProfile® Total OXPHOS Rodent WB Antibody Cocktail (MS604, MitoSciences).
P drp1 ser637
Manufactured by Cell Signaling Technology
Sourced in United States
P-DRP1 (Ser637) is a primary antibody that detects phosphorylation of dynamin-related protein 1 (DRP1) at serine 637. DRP1 is a GTPase that plays a key role in mitochondrial fission. Phosphorylation of DRP1 at serine 637 inhibits its GTPase activity and mitochondrial fission.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
P drp1 ser616, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
P mst1 2, by Cell Signaling Technology (1 mentions)
Nf κb2, by Cell Signaling Technology (1 mentions)
Actin 8h10d10, by Cell Signaling Technology (1 mentions)
Mitoprofile total oxphos rodent wb antibody cocktail, by Abcam (1 mentions)
β actin, by AbFrontier (1 mentions)
Calcineurin, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Multi gauge version 3, by Fujifilm (1 mentions)
Ubiquitin, by Novus Biologicals (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Vdac1, by Novus Biologicals (1 mentions)
P62 sqstm1, by Novus Biologicals (1 mentions)
Pink1, by Santa Cruz Biotechnology (1 mentions)
10 protocols using p drp1 ser637
1
Comprehensive Molecular Profiling of Cellular Signaling
2
Mitochondrial Dynamics and Apoptosis Assay
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Whole protein lysates were extracted from cells using PRO-PREP Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea). Cytoplasmic and mitochondrial fractionation was performed using a mitochondrial isolation kit for cultured cell (Thermo Scientific, MA, USA) according to the manufacturer’s protocol. Proteins were separated by 8–12% SDS-PAGE and then transferred onto nitrocellulose membranes (Pall, FL, USA). Membranes were incubated with antibodies against dynamin-related protein 1 (Drp1) (Cat#sc-32898), Mfn1 (Cat#sc-50330), Mfn2 (Cat#sc-50331) (Santa Cruz, CA, USA), p-Drp1 (Ser637) (Cat#4897S), cleaved caspase-3 (Cat#9661s), calcineurin (Cat#2614S; Cell Signaling, Danvers, MA, USA), Prx5 (Cat#LF-PA0210), and β-actin (Cat#LF-PA0207) (Abfrontier, Korea). We used horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgGs (Thermo Scientific) as secondary antibodies. Protein bands were visualized with Clarity Western ECL Substrate (Bio-Rad, CA, USA), and band intensities were analyzed with Multi Gauge version 3.0 software (Fujifilm, Japan).
3
Protein Expression Analysis in TH1 Cells and Renal Cortex
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The whole cell lysates, cytosol fraction lysates, or mitochondrial fraction lysates were prepared from TH1 cells and renal cortex tissue. Protein concentration was determined by employing the BCA method, and 30 μg of protein per sample was loaded for western blotting. Electrophoresis in 8–12% sodium dodecyl sulfate-polyacrylamide gel was followed by a transfer to polyvinylidene difluoride membranes. The blots were washed with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20) and blocked with 5% skim milk for 1 h at room temperature. Subsequently, we incubated the blot with the appropriate primary antibodies: against PINK1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-517353), SIAH3 (Novus, Littleton, CT, USA, NBP2-83524), LC3B (Novus, NB100-2220), P62/SQSTM1 (Novus, NBP1-48320), p-DRP1 (Ser637) (Cell Signaling Technology, #4867S), DRP1 (Santa Cruz Biotechnology, sc-271583), MFN1 (Santa Cruz Biotechnology, sc-166644), OPA1 (Novus, NBP1-71656), ubiquitin (Novus, NB300-130), VDAC1 (Novus, NB100-695), α-tubulin (Santa Cruz Biotechnology, sc-8035), and β-actin (Santa Cruz Biotechnology, sc-47778). Then, the membranes were washed again and incubated for detection with either goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (Cell signaling, Danvers, MA, USA). The bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
4
Mitochondrial Dynamics Regulation Analysis
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IP/WB analysis was carried out as described previously.6 The antibodies against the following molecules were used for the analyses: Sirt6 (1:500; Abcam; #ab62739), Drp1 (1:1000; Abcam; #ab56788), mitofusin2 (Mfn2) (1:1000; Abcam; ab56889), mitochondrial fission protein 1 (Fis1) (1:1000; Genetex; GTX111010), p‐Drp1‐Ser616 (1:1000; Cell Signalling Technology; #3455), optic atrophy 1 (OPA1) (1:1000; ImmunoWay; YN2976), and p‐Drp1‐Ser637 (1:1000, Cell Signalling Technology; #4867).
5
Quantitative Western Blotting of Mitochondrial Dynamics
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Western immunoblotting was performed as described previously [3 (link)]. Briefly, total protein samples were separated by SDS-PAGE and then transferred to PVDF membranes (Sigma, USA), which were blocked with 5% nonfat milk for 1 h and incubated overnight at 4°C with the primary antibody (Nrf2, #GTX103322; Keap1, Novus, USA, #NBP1-83106; optic atrophy-1 (Opa1), ImmunoWay, USA, #YN2976; mitofusins2 (Mfn2), Abcam, USA, #ab124773; dynamin-related protein-1 (Drp1), Abcam, #ab56788; p-Drp1-Ser637, Cell Signaling Technology, USA, #4867; fission 1 (Fis1), GeneTex, #GTX111010; and GAPDH, Proteintech, China, #600004-1). HRP-labeled goat anti-rabbit/mouse IgG (H+L) antibody (Antgene, China) was used as the secondary antibody. The proteins were detected by a Bio-Rad imaging system.
6
Mitochondrial Dynamics Protein Analysis
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Protein samples were homogenized in ice-cold RIPA lysis buffer with phosphatase inhibitor (Sigma-Aldrich, #04906845001) and protease inhibitor (ThermoFisher, #87785). Lysed cells were incubated on ice for 20 min and centrifuged at 13,500g for 20 min at 4 °C. Collected supernatants were subjected to a Pierce BCA protein assay (ThermoFisher, #23225) to quantify protein concentration. The lysate samples were separated via Novex 4–20% Tris-Glycine gel and transferred to an immobilon-P membrane (Millipore). The membrane was visualized by standard enhanced chemiluminescence. Antibodies were from the following sources: VDAC1 (abcam, ab15895), Histone H3 (abcam, ab1791), MFN1 (abcam, ab57602), MFN2 (Santa Cruz, sc-100560), β-actin (Sigma-Aldrich, A1978), DRP1 (BD Biosciences, 610296), pDRP1 Ser637 (Cell signaling technology, #6319), Tom20 (Cell signaling technology, #42406), TFAM (Novus, H00007019-B01P), p53 (Santa Cruz, sc-126), PINK1 (Cell signaling technology, #6964), LC3B (Santa Cruz, sc-16755), p21 (Cell signaling technology, #2947) and Polymerase gamma (Novus, #NBP1-52300), CHCHD4 (ProteinTech, #21090-1-AP), PGC-1α (Novus, P1-04676).
7
Mitochondrial Dynamics in Parkinson's Disease
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Sinapic acid (SA, Cat# D7927), MPTP (Cat# M0896) and MPP+ (Cat# D048) were obtained from Sigma–Aldrich (St. Louis, MO, USA). SR8278 (SR, Cat# 4463) and GSK4112 (GSK, Cat# 3663) were purchased from Tocris Bioscience (Bristol, UK). DMEM and FBS were obtained from HyClone (Logan, UT, USA). Trypsin-EDTA and a mixture of penicillin and streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA). Rabbit anti-TH (Cat# 2792S), REV-ERB α (Cat# 13418S), p-Drp1 Ser616 (Cat# 3455S), p-Drp1 Ser637 (Cat# 4867S), and GAPDH (Cat# 2118S) were purchased from Cell Signaling Technology Inc (Boston, MA, USA). Anti-rabbit (Cat# 7074S) and mouse (Cat# 7076S) horseradish peroxidase (HRP)-linked IgG antibodies were also obtained from Cell Signaling Technology Inc. The mouse anti-Drp1 antibody (Cat# sc-271583) was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). The rabbit anti-OPA1 antibody (Cat# ab157457) and MFN2 (Cat# ab56889) were purchased from Abcam (Cambridge, UK).
8
Western Blot Analysis of Mitochondrial Dynamics Proteins
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After treatment, HT22 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, USA) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Shanghai, China). Total protein quantification and western blot procedures were performed routinely [34 (link)]. Then, equal amounts of proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with blocking buffer, 5% nonfat milk, or 5% BSA (for phosphoproteins) for 1.5 h. Then, the membranes were incubated with primary antibodies against Drp-1, Mfn1, Mfn2, Fis1, β-actin (Proteintech, Wuhan, China), p-Drp1 (Ser637), and Opa1 (Cell Signaling Technology, CST, USA). After that, the membranes were incubated with corresponding secondary antibodies, namely, goat anti-rabbit IgG or goat anti-mouse IgG (Proteintech, Wuhan, China). The proteins were detected using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, USA) and visualized by a chemiluminescence imaging system (Azure 300, Azure Biosystems, USA).
9
Characterization of Mitochondrial Dynamics Regulators
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Primary antibodies specific for p-PRAS40 (Thr246), DRP1, p-DRP1 (Ser616), and p-DRP1 (Ser637) were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA) and specific for mTOR, PRAS40, E-cad, N-cad, and Vimentin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and specific for α-tubulin and p-mTOR (Ser2448) were purchased from Abcam (Abcam, Cambridge, MA, USA) and specific for DYRK3 was purchased from Thermo Fisher Scientific (Thermo Fisher Scientific, Cleveland, OH, USA). Secondary antibodies specific for mouse IgG and rabbit IgG were purchased from Enzo Life Sciences (Enzo Life Sciences, Ann Arbor, MI, USA). Eagle’s Minimum Essential Medium (MEM), Hanks’ Balanced Salt solution (HBSS), Phosphate Buffered Saline (PBS), and fetal bovine serum (FBS) were acquired from WelGENE Inc (WelGENE Inc., Daegu, Korea). Penicillin, streptomycin, recombinant human EGF protein, Trizol, MitoTrackerTM Green FM and MitoSOXTM Red were obtained from Thermo Fisher Scientific (Thermo Fisher Scientific, Cleveland, OH, USA). SiRNAs specific for human DYRK3 and control siRNA were purchased from Bioneer (Bioneer, Daejeon, Korea).
10
Immunoblotting Analysis of Cellular Proteins
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Immunoblotting was performed as described previously (43 (link)). Briefly, protein samples were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane probed with the following antibodies: Drp1 (BD Biosciences, 610296), CD144 (Invitrogen, 14-1441-81), p–Drp1 Ser637 (Cell Signaling Technology, 6319), β-actin (Sigma-Aldrich, A1978), VCAM-1 (Santa Cruz Biotechnology Inc., sc-13160), CD31 (MilliporeSigma, MAB1398Z), PDK1 (Santa Cruz Biotechnology Inc., sc-515944), HK2 (Santa Cruz Biotechnology Inc., sc-374091), T-eNOS (BD Biosciences, 610296), OPA1 (BD Biosciences, 612606), Mfn2 (Santa Cruz Biotechnology Inc., sc-100560), Fis1 (Sigma-Aldrich, HPA017430), and α-tubulin (Sigma-Aldrich, T9026). Detailed methods are provided in the Supplemental Methods .
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