Metformin was purchased from Cayman Chemical (Michigan, USA). Compound C was purchased from Sigma (USA.). A potentiometric fluorescent dye tetramethylrhodamine ethyl ester (TMRE) was purchased from Life Technology (USA.); iTaq™ universal SYBR® Green supermix was purchased from Bio-Rad (USA). Antibodies were obtained from the following commercial sources: mouse actin (Sigma); total mouse AMPK (Cell Signaling); phospho-AMPK (Cell Signaling); anti-Tomm-20 (Santa Cruz Biotechnology). Protein assay BCA kits were purchased from Thermo Scientific. Cell culture medium and supplements including Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), Penicillin/Streptomycin, G418, and GlutaMAX were purchased from Life Technology (USA). All other chemicals and reagents were commercially available and of the highest grade. Dominant Negative (DN) myc-tag labeled AMPKα cDNA construct in pcDNA3 (Invitrogen) plasmid was generously provided by Dr. David Carling MRC Clinical Sciences Centre, Imperial College, London, UK), the plasmid was amplified and cDNAs were extracted and provided by Dr. Edward Gabrielson lab at Johns Hopkins University School of Medicine.
Anti tomm20
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-TOMM20 is a laboratory product used for the detection and study of TOMM20, a protein that is a component of the translocase of the outer mitochondrial membrane. It can be used in various research applications involving the analysis of mitochondrial function and structure.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Anti lc3b, by Merck Group (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti ha, by Cell Signaling Technology (2 mentions)
Anti tubulin, by Merck Group (2 mentions)
Anti mavs, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Tetramethylrhodamine ethyl ester tmre, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Compound c, by Merck Group (1 mentions)
Phospho ampk, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Metformin, by Cayman Chemical (1 mentions)
3 well chamber slides, by Ibidi (1 mentions)
Anti β tubulin, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
710 confocal microscope, by Zeiss (1 mentions)
21 protocols using anti tomm20
1
AMPK Activation Pathway Analysis
2
Immunostaining of Dental Pulp Stem Cells
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Dental pulp stem cell (DPSC) were grown and differentiated on 3-well chamber slides (Ibidi, Planegg, Germany) coated with poly-D-lysine. Cells were fixed using a 4% paraformaldehyde solution for 10 min. Once fixed, cells were blocked and permeabilized using PBS with 1% BSA, 10% FBS, and 0.3% Triton X-100 for 1 h. Primary antibodies were diluted to 1:500 for anti-Beta Tubulin (Millipore, ab9354) and anti-TOMM20 (Santa Cruz, sc-17764) in the blocking solution and incubated overnight at 2–8°C with agitation. After overnight incubation, the slides were washed 3x with PBS-T for 10 min before the secondary antibodies, Goat anti-mouse Alexa Fluor 488 (Life Technologies, A11029) and Goat anti-chicken Alexa Fluor 594 (Life Technologies, A11042) were added at a 1:1000 dilution. Slides were incubated at room temperature for 1 h and then washed again 3× for 10 min each. Finally, Prolong Gold Antifade with DAPI (Fisher Scientific, Waltham, MA) was applied for mounting. Slides were imaged on a Zeiss 710 confocal microscope at 63× magnification using Z-stacking to image the entire neuron via ZEN software (Black Edition).
3
Protein Localization and Autophagy Markers
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The following primary antibodies were used: anti-GAPDH (Sigma-Aldrich, G9545; 1:5000) anti-TOMM20 (Santa Cruz Biotechnology, SC-11415; 1:500), anti-FIS1 (Proteintech, 10956–1-1ap; 1:100), anti-ATP5F1A/ATP5A (Abcam, Ab14748; 1:200), anti-PDHA1 (Abcam, ab110330; 1:200), anti-ATG7 (Cell Signaling Technology, 8558; 1:1000), anti-RAB9A (Cell Signaling Technology, 5118; 1:1000), anti-ULK1 (Cell Signaling Technology, 8054; 1:1000), anti-RAB5A (Cell Signaling Technology, C8B1; 1:1000), anti-RAB7 (ERP7589; Abcam, ab137029; 1:1000), anti-LC3B (Sigma-Aldrich, L7543;1:200). Alexa Fluor 647‐conjugated goat anti-rabbit and anti-mouse IgG (Invitrogen, A21244 and A32728; 1:500) were used as secondary antibodies.
4
Immunoprecipitation and Immunoblot Analysis
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The primary antibodies used include anti-LC3B, anti-beclin1 (Novus Biologicals, Littleton, CO, USA), anti-caveolin1 (Abcam, Cambridge, UK), anti-TIMM23, anti-phospho-caveolin1 (BD Bioscience, San Jose, CA, USA), anti-HA, anti-Vps34 (Cell Signaling Technology, Danvers, MA, USA), anti-tubulin, anti-Flag (Sigma-Aldrich), anti-p62 (Abnova, Taiwan) and anti-TOMM20 (Santa Cruz Biotechnology, Dallas, TX, USA). Immunoprecipitation assays were performed as previously described;6 (link) briefly, cells were lyzed with modified RIPA buffer (10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1% CHAPS). After pull-down with the appropriate antibodies, same amounts of protein were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto the polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Immunoblot analysis was then performed and visualized by the enhanced chemiluminescence method.
5
Immunofluorescence and Western Blot Protocols
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The following primary antibodies were used in this study: anti-TOMM20 (Santa Cruz Biotechnology; sc-17764, IF 1:1,000), anti-mNG (Chromotek, 32f6; IF 1:500), anti-GM130 (Cell Signaling Technology, #12480; IF 1:1,000), anti-HNRNPA1 (Santa Cruz Biotechnology, sc-32301; WB 1:500), anti-mNG (Cell Signaling Technology, #53061, WB 1:100), and anti-HSP90 (BD Biosciences, 610419; WB 1:5,000). The following secondary antibodies were used: donkey anti-mouse IgG Alexa Fluor 555 (Invitrogen, A32773; IF 1:500), donkey anti-mouse IgG Alexa Fluor 647 (Invitrogen, A32787; IF 1:500), donkey anti-rabbit IgG Alexa Fluor 647 (Invitrogen, A32795; IF 1:500), anti-mouse IgG HRP (Promega, WB 1:10,000), and anti-rabbit IgG HRP (Promega, WB 1:10,000).
6
Investigating IGF-1 Signaling in C2C12 Myoblasts
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C2C12 myoblasts were purchased from ATCC (Shanghai, China). Dulbecco modified Eagle’s medium (DMEM), penicillin/streptomycin (P/S), fetal bovine serum (FBS), and horse serum (HS) were purchased from Thermo Scientific (Waltham, MA, USA). IGF-1, BMS754807 (BMS), 3-MA, and chloroquine (CQ) were purchased from MedChemExpress (Shanghai, China). Antibodies such as anti-phospho-IGF-1R, anti-IGF-1R, anti-phospho-AKT, anti-AKT, anti-phospho-mTOR, and anti-mTOR were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-BNIP3, anti-myosin heavy chain (MHC), anti-Myogenin (MyoG), and anti-TOMM20 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies such as anti-α-tubulin, anti-GAPDH, anti-PGC-1α, anti-p62, and anti-LC3 were obtained from Protientech (Wuhan, China).
7
Immunostaining Them1 in Adipose Tissue
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Differentiated iBAs were fixed, as above, and then stained for perilipin (anti-Plin1) to identify LD, Tomm20 (anti-Tomm20) to detect mitochondria, or anti-EGFP to visualize the plasmid-transfected Them1-EGFP. For staining Them1 in paraffin sections of BAT, tissues were treated with Image-iT™ FX Signal Enhancer (Thermo Fisher Scientific, Hampton, NH) after antigen retrieval with citrate buffer, pH 6.0. The sections were stained with anti-Them1 antibody, anti-Plin1, and Hoechst 33342 to identify nuclei.
Details of the antibodies: affinity purified rabbit anti-Them111 (link) 1:250 for immunostaining or 1:1000 for immunoblots (see Supplementary Fig.5 ); anti-Tomm20, Santa Cruz Biotech (Dallas TX, USA), rabbit, sc-11415, 1:200; anti-Plin1, Fitzgerald (Acton MA, USA), guinea pig, 20R-PP004,1:500; anti-EGFP, Novus Biologicals (Littleton CO, USA), goat, NB100-1678, 1:500; anti-actin, Abcam (Cambridge, MA, USA), mouse, Ab3280,1:1000. Secondary antibodies labeled with HRP, AlexaFluor 488, 647, or Cy3 were purchased from Jackson ImmunoResearch (West Grove PA, USA).
Details of the antibodies: affinity purified rabbit anti-Them111 (link) 1:250 for immunostaining or 1:1000 for immunoblots (see Supplementary Fig.
8
Immunofluorescence and Immunoblotting Reagents
9
Lysosomal and Autophagy Protein Detection
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Cell pellets were extracted using SDS extraction buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% DOC, 1% NP-40, and 0.1% SDS) or 1% Triton X-100 lysis buffer (containing 10% glycerol, 150 mM NaCl, 25 mM Hepes pH 7.4, 1 mM EDTA, 1.5 mM MgCl2, proteinase inhibitor cocktail) and gels were blotted onto nitrocellulose membranes. Antibodies used for immunoblotting were as follows: anti-LAMP (Lysosome-associated membrane glycoprotein)-1 (1:1000; Santa Cruz), anti-LAMP-2 (1:1000; Santa Cruz), anti-GBA (Lysosomal acid glucosylceramidase; 1:1000; Abcam), anti-GAA (Lysosomal alpha-glucosidase; Invitrogen; 1:1000), anti-TOMM20 (translocase of the outer mitochondrial membrane 20; 1:1000; Santa Cruz), anti-LC3A/B (microtubule-associated protein 1 light chain 3; 1:500; Cell Signaling), anti-Beclin 1 (1:1000, Cell Signaling), anti-Sequestosome1 (p62; 1:1000; Cell Signaling), anti-GPX4 (Glutathione peroxidase 4; 1:1300; Abcam), anti-ferritin heavy chain (FTH, 1:600; Cell Signaling), anti-NCOA4 (1:500; Bethyl Laboratories), and anti-β-actin (1:1,000,000; Sigma).
10
Protein Extraction and Analysis Protocol
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Cells were rinsed in phosphate-buffered saline (PBS) on ice and lysed in RIPA (Radioimmunoprecipitation assay buffer) buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich, P8340, Milan, Italy), Na4VO3 0.1 mM (Sigma-Aldrich, S6508, Milan, Italy), NaF 1 mM (Sigma-Aldrich, S7920, Milan, Italy) and β-Glycerophosphate 5 mM (Sigma-Aldrich, G6376, Milan, Italy). Cell extracts were centrifuged at 15,000× g for 10 min at 4 °C. Protein concentrations were determined with the Bio-Rad Protein Assay Kit (Bio-Rad, 5000001, Milan, Italy). Cell extracts were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Primary antibodies used were: anti-SOD2 (Enzo-Lifesciences, Milan, Italy), anti-TOMM20 (Santa Cruz Biotechnology, Milan, Italy), anti-PRKN (Cell signalling, Milan, Italy), anti-VCL (Santa Cruz Biotechnology, Milan, Italy), anti-Ub (Santa Cruz Biotechnology, Milan, Italy), anti-Ub (KK2, Enzo-Lifesciences, Milan, Italy), anti-HSP60 (Santa Cruz Biotechnology, Milan, Italy). All uncropped images are illustrated in Supplementary Figure S1 ).
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