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13 protocols using ab110334

1

Multicolor Immunofluorescence Labeling

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Primary antisera used were rabbit anti-Vasa (R. Lehmann laboratory), mouse monoclonal anti-Hts (1B1, DSHB)39 , mouse monoclonal anti-Orb (4H8, DSHB)40 , mouse monoclonal anti-HA (abcam ab130275), chicken anti-GFP (Aves Labs, GFP-1020), mouse monoclonal anti-ATP5A [15H4C4] (abcam ab14748), mouse anti-PDH E1 alpha (Abcam, ab110334) and rabbit anti-phoshpo-PDH E1alpha (S293) (Millipore, AP1062). Secondary antibodies were DyLight™ 405 donkey anti-rabbit, DyLight™ 405 donkey anti-mouse, Cy3 donkey anti-mouse, all from Jackson ImmunoResearch, and Alexa fluor 488 goat anti-chicken from ThermoFisher Scientific.
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2

Western Blot Analysis of Cellular Proteins

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The cells were lysed using RIPA buffer (150 mM NaCl, 50 mM Tris-HCl at pH 7.4, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate and 1% NP-40) mixed with a protease and phosphatase inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and phenylmethylsulfonyl fluoride (PMSF) (Biosharp, Hefei, China) for 15 min on ice. The proteins were subjected to western blotting according to standard protocols. Antibodies were as follows: SIRT2 (HPA011165,Sigma), SIRT2 (s8447, Sigma), cMYC (ab32072, Abcam, Cambridge, UK), p293-PDHA1 (ab92696, Abcam), GAPDH (ab128915, Abcam), cleaved-caspase3 (9664s, CST, Danvers, MA, USA), cleaved-PARP (5625s, CST), PDHA1 (ab110334, Abcam), PHGDH (14719-1-AP, Proteintech, Chicago, IL, USA), PSAT1 (10501-1-AP, Proteintech), PSPH (14513-1-AP, Proteintech), HRP-conjugated anti-rabbit (7074, CST), and anti-mouse antibodies (7076, CST).
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3

Immunoblot Analysis of Myocardial Proteins

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Protein was extracted from frozen RV tissues and 40–60 μg of total protein was loaded to SDS-PAGE gel for immunoblot analysis, as previously described31 (link) (also see Supplemental methods). The following antibodies were used: PDH E1-alpha subunit (phospho S293) (P-PDH) (ab177461; Abcam, Cambridge, MA, USA), PDH E1-α subunit (ab110334; Abcam, Cambridge, MA, USA), PKM1 (NBP2–14833; Novus Biologicals, Littleton, CO, USA), PKM2 (60268–1-Ig; Proteintech, Rosemont, IL, USA), and vinculin (V9131; Sigma, St. Louis, MO, USA). vinculin was used as loading control.
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4

Immunofluorescence Analysis of Pancreatic Islet Cells

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Tissue samples were cut into sections and processed as aforementioned. Slides were incubated with mouse anti-PDHA1 (1:200; cat. no. ab110334; Abcam) and rabbit anti-insulin (1:200; cat. no. ab63820; Abcam) overnight at 4°C in a wet box. The slides were washed for 3 min three times and incubated in the dark with the corresponding diluted secondary antibodies [1:500; cat. no. ab150077 (Alexa Fluor® 488-conjugated goat anti-rabbit IgG) and ab150116 (Alexa Fluor 594-conjugated goat anti-mouse IgG; Abcam) at 37°C for 2 h. The secondary antibody solution was discarded and the slides were washed three times. Slides were incubated with DAPI (1drop: 1 ml, ~1 min at room temperature. Images were captured under a laser confocal microscope and 5 visual fields containing pancreatic islet cells were selected under a the microscope (magnification, ×400); the number of PDHA1 positive cells and total number of cells in each visual field were counted by Image-Pro Plus 6.0 software (Media Cybernetics, Inc.). The rate of PDHA1 positive cells in islet cells was calculated.
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5

Quantitative Mitochondrial Protein Analysis

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For each specimen, four thoraxes were dissected and homogenized by dounce tissue grinder in RIPA buffer containing protease inhibitor (cOmpleteTM, Roche). Cellular debris were removed by centrifugation at 4 °C, 14000× g for 20 min. The supernatants were collected and the protein concentrations were determined by Pierce protein assay (Pierce 660 nm Protein Assay Reagent, ThermoScientific). 0.6 μg/well of proteins were loaded for SDS-PAGE and western-blot analysis.
The primary antibodies and the dilutions were as follows: mouse anti-ATP5A (50000x, abcam ab14748), mouse anti-Cytochrome C (10000x, abcam ab13575), mouse anti-PDHA1 (1000x, abcam ab110334), rabbit anti-SOD2 (10000x, abcam ab13534) and rabbit anti-alpha Tubulin (10000x, abcam ab18251). The secondary antibodies and the dilutions were as follows: anti-mouse IgG-HRP (2000x, Invitrogen 62–6520), or anti-rabbit IgG-HRP (5000x, abcam ab97051). The signals were developed by chemiluminescent HRP substrate (ImmobilonTM Western, Millipore) and recorded by a digital multispectral imaging system (BioSpectrum, UVP).
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6

Phosphorylated PDH-E1α and Glucose Transporters

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Cells were lysed in a low detergent buffer (1% Triton, 0.1% SDS) for one hour with protease and phosphatase inhibitors (Sigma-Aldrich). Nitrocellulose membranes were hybridized with anti-phospho S232-PDH-E1α (Millipore AP1063), total PDH-E1α antibodies (Abcam ab110334), Glut1 rabbit monoclonal (abcam, ab115730), Glut3 rabbit polyclonal (abcam, ab15311), actin (Cell Signaling, 4970S), cMyc (Cell Signaling, 179) or Hif1α (Cayman Chemicals 10006421).
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7

Metabolic Regulation in Hypoxia

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HIF-1α antibody 1:1000 (abcam reference #ab51608), GLUT1 antibody 1:1000 (Merk Millipore reference #07-1401), PDK1 antibody 1:1000 (abcam reference #ab110025), HIF-1α OH-P402 1:1000 (abcam reference #ab72775), Beta-actin antibody 1:10000 (cell signaling, Cat. #4967), Pyruvate dehydrogenase E1-alpha subunit 1:2000 (abcam reference #ab ab110334) and Pyruvate dehydrogenase E1-alpha subunit (phospho S293) 1:1000 (abcam reference #ab177461) were used for western blot. GLUT1 and PDK1 were used for Immunohistochemistry assays.
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8

Western Blot Analysis of Metabolic Proteins

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The cells were lysed using lysis buffer (Biosharp) to obtain total protein. Next, loading buffer containing 5% 2‐mercaptoethanol was mixed with the lysate. The proteins were denatured at 100°C for 10 minutes before being separated on 8%‐12% sodium dodecyl sulfate‐polyacrylamide gels. After transfer to PVDF membranes (Millipore), the membranes were incubated for 2 hours at room temperature in TBST containing 5% skim milk. Afterward, membranes were incubated with the corresponding primary antibodies overnight at 4°C according to the manufacturer's instructions. After treatment with the appropriate horseradish peroxidase (HRP)‐conjugated secondary antibodies, the membranes were incubated with ECL western blotting reagents (Millipore). The following antibodies were used: SIRT3 (s4072; Sigma), cMYC (ab32072; Abcam), HIF1α (36169; CST, Danvers, MA, USA), PDK1 (5662; CST), p‐PDHA1 (ab92696; Abcam), GAPDH (ab128915; Abcam), Actin (A5441; Sigma), PDHA1 (ab110334; Abcam), GLUT1 (ab115730; Abcam), Anti‐mouse IgG (7076; CST), and Anti‐rabbit IgG (7074; CST).
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9

Western Blot Analysis of Mitochondrial Proteins

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Whole flies or dissected fly tissue were homogenised in 2x Laemmli loading sample buffer (100 mM Tris pH 6.8, 20% glycerol, 4% SDS; Bio-Rad) containing 50 mM DTT. Extracts were cleared by centrifugation and approximately 20–40 μg of protein extract was loaded per lane on a polyacrylamide gel. Proteins were separated and transferred to a nitrocellulose membrane. The following antibodies were used at the indicated dilutions: β-actin (Abcam, ab8227; 1:4000), Atg8 (a generous gift from Katja Köhler; 1:2000; [80 (link)], PDH (Abcam, Ab110334), NDUFS3 (Abcam, ab14711), GAPDH (GeneTex, GTX100118), cytochrome C (BioLegend, 612503, clone 7H8.2C12), SDHB (Abcam, ab14714), ATP5A (Abcam, ab14748), VDAC (Abcam, ab14734). The HRP-conjugated anti-rabbit secondary (Abcam, ab6721; 1:12000) was used. Blots were developed using the ECL detection system (GE, Amersham), and analysed using FIJI software (US National Institutes of Health). To quantify total protein, we used TGX stain-free gels from Bio-Rad (567–8123 or 567–8124) according to the manufacturer’s instructions. For hydroxychloroquine (HCQ) treatment, HCQ was dissolved in water to give a 10M stock. The stock was then added to standard SYA fly food to give a final concentration of 30mM. Flies were pretreated 24h with HCQ before western blot analysis.
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10

Immunogold Labeling of Cellular Organelles

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Thin sections placed on nickel grids were blocked with 10% BSA in PBS for 20 min, and incubated with primary antibodies in incubation buffer (1% BSA in PBS) for 2 hr. Grids were subsequently washed with incubation buffer three times (10 min each). Secondary antibodies, goat anti-mouse IgG (EM.GMHL15, BB International) and protein A (EM.PAG15, BB International) conjugated to 15 nm gold particles, were used against the primary antibodies from mouse and rabbit respectively. Secondary antibodies at 20-fold dilution were applied and samples were incubated for 1 hr. After washing with PBS, the immune-complexes were fixed with 1% glutaraldehyde in PBS and washed three times with distilled water. The specimens were inspected by TEM operating at 120 kV (FEI Tecnai G2 TF20 Super TWIN).
The primary antibodies and applied dilution factors are listed as follows: mouse anti-dsDNA (500x, abcam ab27156), mouse anti-ATP5A (500x, abcam ab14748), mouse anti-Cytochrome C (8000x, abcam ab13575), mouse anti-PDHA1 (500x, abcam ab110334), mouse anti-Ubiquitin (1000x, abcam ab7254), mouse anti-DNA-RNA hybrid (500x, kerafast ENH001), and rabbit anti-SOD2 (500x, abcam ab13534).
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