Protein concentration of subcellular fractions isolate was measured by a Pierce BCA Method (Thermo Fisher Scientific #23227). 50 μg of protein were loaded onto 15% PAGE-SDS gels and electrophoresis was performed at a constant voltage of 100 V at 4°C. Transference of proteins was performed at 15 V overnight at 4°C. Antibodies used were: autophagosomal marker, anti-LC3 (MBL #PM036); lysosomal markers, anti-LAMP1 (DSHB #1D4B); anti-Cathepsin D (Abcam #ab75852); and mitochondrial markers, anti-VDAC (Cell Signaling #4661); anti-TOM20 (Cell Signaling #13929); anti-Cytochrome C (BD Pharmingen #556432). HRP-conjugated secondary antibodies were from Sigma.
Anti tom20
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-TOM20 is a primary antibody that recognizes the TOM20 protein, a component of the translocase of the outer membrane (TOM) complex in mitochondria. TOM20 functions as a receptor for the import of nuclear-encoded mitochondrial proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Anti bax, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti pink1, by Cell Signaling Technology (2 mentions)
Las x software, by Leica (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Anti mfn2, by Cell Signaling Technology (2 mentions)
Anti drp1, by Cell Signaling Technology (2 mentions)
Anti cytochrome c, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti vinculin, by Cell Signaling Technology (2 mentions)
Anti lc3, by Cell Signaling Technology (2 mentions)
Ab152, by Merck Group (2 mentions)
Anti ampk, by Cell Signaling Technology (2 mentions)
Mitosox red, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Merck Group (1 mentions)
Anti cytochrome c, by BD (1 mentions)
Anti cathepsin d, by Abcam (1 mentions)
Pierce bca method, by Thermo Fisher Scientific (1 mentions)
Anti vdac, by Cell Signaling Technology (1 mentions)
26 protocols using anti tom20
1
Subcellular Fraction Protein Analysis
2
Molecular Profiling of Mitochondrial Dynamics
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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).
3
Protein analysis of muscle tissues
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For the protein extracts, myotubes or TA muscles were homogenized in a radioimmunoprecipitation assay (RIPA) buffer with 1 mM of a cocktail of protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) and 1 mM of phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA). Proteins were subjected to SDS-PAGE, transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Waltham, MA, USA), and probed with anti-OXPHOS (1:1000; Abcam, Cambridge, MA, USA), anti-PGC-1α (1:1000, Cell Signaling, Danvers, MA, USA), anti-PINK-1 (1:1000, Cell Signaling, Danvers, MA, USA), anti-LC3B (1:1000, Cell Signaling, Danvers, MA, USA), anti-TOM20 (1:1000, Cell Signaling, Danvers, MA, USA), anti-GAPDH (1:2000; Santa Cruz, Dallas, TX, USA) and anti-β-actin (1:1000; Abcam, Cambridge, MA, USA) antibodies. All immunoreactions were visualized by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA). Images were acquired using the Fotodyne FOTO/Analyst Luminary Workstation Systems (Fisher Scientific, St. Waltham, MA, USA). Densitometry analysis was determined by scanning immunoreactive bands. Intensity values were obtained for further normalization against the control group using ImageJ software (National Institutes of Health [NIH], Bethesda, MD, USA).
4
Mitochondrial, Lysosomal, and Nuclear Staining
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MDMs were seeded at a density of 20,000 cells/well. Then mitochondria were stained using anti-MTCO2 (Abcam) or anti-TOM20 (Cell Signaling Technology). Lysosomes were stained with anti-LAMP1 (Cell Signaling Technology), and nuclei were stained with DAPI (Acros Organics). Fluorescence microscopy was conducted with FV10-ASW4.1 viewer and Leica SP5 DM microscope and analyzed with LASX software (Leica).
5
Apoptosis Regulation by BCL-2 Inhibitors
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The following anti-human antibodies were used: mouse anti-SQSTM1/p62 (sc-28359) and rabbit (sc-130656) or mouse (sc-47778) anti-Actin, from Santa Cruz (Santa Cruz, CA, USA), Mouse anti-LC3 (5F10, 0231-100) from Nanotools (Munich, Germany), P62, and anti-BCL-2 (Santa Cruz, CA, USA), anti-TOM20 and anti-VMP1 were obtained from cell signalling (Leiden, the Netherlands), Hydroxychloroquine (HCQ), was obtained from Sigma-Aldrich. Venetoclax/ABT-199 (BCL-2 inhibitor, Selleckchem Munich, Germany), S63845 (MCL-1 inhibitor) was obtained from APExBIO (Boston, MA, USA). The pan caspase inhibitor ZVAD-FMK was obtained from (Enzo Life Sciences, Bruxelles, Belgium).
6
Immunofluorescence Microscopy of Mitochondria
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Immortalised HSFs were cultured on 1.5 mm cover slips and fixed for 20 min at room temperature using 3% paraformaldehyde. Permeabilisation and blocking was performed using a combination of 0.1% saponin (Merck), 1% BSA (Merck) in PBS for 1 h. Primary antibody anti-TOM20 (42406, Cell Signalling) was diluted 1:200 in PBS with 0.1% saponine and 1% BSA and incubated overnight. After washing, cells were incubated for 1 h with secondary antibody Alexa Fluor 568 (Goat anti-Mouse IgG (H + L) antibody Alexa Fluor 568, A-11031, Thermo Fisher, or Goat anti-Rabbit IgG (H + L) antibody Alexa Fluor 568, A-11036, Thermo Fisher) at a 1:1000 dilution. Cells were mounted with ProLong Gold Antifade Mountant and 0.01 μg/ml DAPI (Merck). Images were acquired using a Nikon Ti-E inverted point scanning confocal microscope A1R Si.
7
Mitochondrial Dynamics Antibody Assay
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The antibodies were as follows: anti-β-Actin (Sigma-Aldrich); anti-TRAP1, anti-Hsp90, anti-COX IV, anti-caspase 3, anti PARP (Santa Cruz Biotechnology); anti-Drp1, anti-Drp1 (Ser616), anti-Mfn1, anti-Mfn2, anti-Tom20, anti-Tim23 (Cell Signaling); MitoProfile antibody cocktail for mitochondrial complexes (Abcam); anti-rabbit or anti-mouse HRP-conjugated secondary antibody (Sigma-Aldrich). All chemicals were from Sigma-Aldrich unless otherwise indicated.
8
Western Blot Analysis of Apoptosis and Mitochondrial Proteins
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The A2780 and SKOV3 cells were lysed with 120 µl of RIPA buffer (Beyotime). Cell lysates were sonicated for 30 sec under ice and lysed at 4°C for 45 min. The cells were lysed by centrifugation at 3,000 × g for 15 min, and the supernatant was used to determine the protein concentration using Bio-Rad kit (Pierce Biotechnology Inc.) and the samples were boiled for 5 min. The cell lysates (10-20 µg) were resolved on 12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 1 h, and incubated with primary antibodies overnight at 4°C. The following day, membranes were washed with PBST and incubated with horseradish peroxidase-conjugated secondary antibodies at 1:2,000 dilution for 1 h at room temperature. After washing the membranes with PBST, immunodetection was performed using ECL reagent (Thermo Fisher Scientific) and visualized using a Syngene Bio Imaging (Synoptics). The primary antibodies used were anti-β-actin (1:1,000 dilution), anti-cleaved caspase-3 (1:1,000 dilution), anti-Bax (1:1,000 dilution), anti-Bcl-2 (1:1,000 dilution), anti-Tom20 (1:1,000 dilution), anti-Drp1 (1:1,000 dilution), anti-cytochrome c (1:1,000 dilution) (Cell Signaling Technology), anti-phospho-DRP1 (Ser637) (1:1,000 dilution), anti-Phospho-AKT (Ser473) (1:2,000 dilution) (Proteintech Group, Inc.).
9
Western Blot Analysis of Muscle Proteins
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Proteins from GC muscles of WT and NRKdKO animals were extracted in RIPA lysis and extraction buffer (Thermo Fisher Scientific, #89901) supplemented with protease (Sigma, #S8820) and phosphatase inhibitor cocktail (Sigma, #4906845001). Protein concentration was determined using a BCA assay (Thermo Fischer Scientific, #23227). Samples were first diluted to 1.5 mg/ml and boiled 5 min in NuPAGE™ LDS Sample Buffer (4X) (Invitrogen, #NP0007), run on 4–12% Bis-Tris Protein gels (Thermo Fischer Scientific, #BN1003), and transferred using the semi dry system from Life Technologies. Membranes were incubated overnight at 4°C with primary antibodies anti-NRK1 and anti-NRK2 (Ratajczak et al., 2016 (link)), anti-NAMPT from Bethyl, OxPhos antibody cocktail from Invitrogen, anti-VDAC, anti-TOM20, anti-IGF1R, anti-PI3K, anti-phospho-Akt (Ser473), anti-Akt, anti-Raptor, anti-FAK, and anti-Vinculin from Cell Signaling. Membranes were then washed and incubated for 1 h with horseradish peroxidase–conjugated donkey anti-rabbit (Jackson ImmunoResearch, #711-035-152, 1/5000). Proteins were visualized with chemiluminescent western blotting substrate (Thermo Fisher Scientific, #32132) using Amersham Hyperfilm™ films. Densitometry analysis was performed using Fiji. Protein levels in each lane were normalized to the levels of Vinculin as a loading control.
10
Western Blot Analysis of Neurodegeneration
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Cell lysates and SNc tissues were homogenized in RIPA lysis buffer, and protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). A 30-μg protein aliquot of each sample was separated using standard SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA). Immunoreactive bands were detected by enhanced chemiluminescence plus detection reagent (Pierce, Rockford, IL) and analyzed using the ImageQuant™ LAS 4000 imaging system (GE Healthcare, Pittsburgh, PA, USA). The following primary antibodies were used: anti-TH (T1299, Sigma, St Louis, MO, USA), anti-p16 (sc-56330, Santa Cruz Biotechnology, USA), anti-β-actin (BM0627, Boster, Pleasanton, CA, USA), anti-LC3 (3868, Cell Signaling Technology, USA), anti-p62 (23214, Cell Signaling Technology, USA), anti-COX IV (4844, Cell Signaling Technology, USA), anti-TOM20 (42406, Cell Signaling Technology, USA), anti-Parkin (ab77924, Abcam, USA), and anti-PINK1 (ab23707, Abcam, USA).
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