Frozen left ventricular (LV) tissue samples (30 mg) were homogenized, resolved by SDS-PAGE, and transferred onto nitrocellulose. The membranes were blocked in 5% fat-free milk for 1 h before being probed with primary antibodies that included: anti–acetyl-lysine (Millipore, AB3879, MilliporeSigma, St. Louis, Missouri), anti–β-hydroxyacyl-CoA-dehydrogenase (βHAD) (ab37673, Abcam, Cambridge, United Kingdom), anti–long-chain-acyl-CoA-dehydrogenase (LCAD) (ab129711, Abcam), anti–pyruvate-dehydrogenase (PDH) (2784, Cell Signaling Technology, Danvers, Massachusetts), and anti–phospho-PDH (PDH ε1a [Ser293]) (Calbiochem, AP1062, MilliporeSigma). The membranes were then incubated with the appropriate secondary antibodies (goat anti-rabbit, catalog sc-2054; goat anti-mouse, catalog sc-2055; and goat anti-chicken, catalog sc-2901; Santa Cruz Biotechnology, Dallas, Texas). Protein bands were visualized with enhanced chemiluminescence and semiquantified via densitometric analysis using the Image J 1.50i software (NIH, Bethesda, Maryland). Tubulin (catalog T6074; MilliporeSigma) acted as the loading control to normalize any variation in protein loading.
Ab129711
Manufactured by Abcam
Sourced in United Kingdom
Ab129711 is a primary antibody that targets the human CD40 antigen. CD40 is a transmembrane glycoprotein that belongs to the tumor necrosis factor receptor superfamily and plays a crucial role in the activation of B cells, dendritic cells, and macrophages. This antibody can be used for various applications, including flow cytometry, immunohistochemistry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Ab37673, by Abcam (3 mentions)
Phospho pdh serine 293 abs204, by Merck Group (2 mentions)
Sc 2055, by Santa Cruz Biotechnology (1 mentions)
Ab3879, by Merck Group (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
T9026, by Merck Group (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
3 protocols using ab129711
1
Quantifying Cardiac Metabolic Enzymes
2
Cardiac Protein Analysis via Western Blot
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Powdered frozen heart samples from mice following isolated working heart perfusion were homogenized in buffer RIPA buffer containing protease and phosphatase inhibitors (Sigma). Protein samples were subsequently prepared and subjected to western blotting protocols as previously described [23 (link)]. Alpha tubulin (T9026, Millipore), aldose reductase (LSC331634, LifeSpan BioSciences), long chain acyl CoA dehydrogenase (LCAD) (ab129711, Abcam), beta-hydroxyacyl CoA dehydrogenase (beta-HAD) (ab37673, Abcam), pyruvate hydrogenase (PDH) (3205S, Cell Signaling), phospho-PDH serine 293 (ABS204, Millipore Sigma), phospho-AKT (4060S, Cell Signaling), AKT (9272S, Cell Signaling), matrix metalloproteases (MMP) 9 (13667S, Cell Signaling), and MMP7 (71031S, Cell Signaling) antibodies were prepared in a 1/1000 dilution in 3% BSA.
3
Cardiac Metabolism Regulation Analysis
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Isolated cardiac myocytes or powdered frozen heart samples from mice following a 16-hr fast and 4-hr refeed (20 mg) were homogenized in buffer containing 50 mM Tris HCl (pH 8 at 4 C), 1 mM ethylenediaminetetraacetic acid, 10% glycerol (w/v), 0.02% Brij-35 (w/ v), 1 mM dithiothreitol, and protease and phosphatase inhibitors (Sigma). Protein samples were subsequently prepared and subjected to western blotting protocols as previously described (Gopal et al., 2017) . PDK4 (AP7041B; Abgent), PDH (3205S; Cell Signaling), phospho-PDH serine 293 (ABS204; Millipore Sigma), phospho-PDH serine 300 (AP1064; Millipore Sigma), phospho-PDH serine 232 (AP1063; Millipore Sigma), long-chain acyl CoA dehydrogenase (ab129711, Abcam), b-hydroxyacyl CoA dehydrogenase (ab37673, Abcam), cleaved caspase 3 (9661S; Cell Signaling), and heat shock protein-90 (610418; BD Biosciences) antibodies were prepared in a 1/1000 dilution in 3% BSA. Immunoblots were visualized using a SuperSignal West Pico chemiluminescence western blot detection kit (Thermo Fisher Scientific) and ImageQuant LAS4000 imager (GE Healthcare), while protein bands were quantified using ImageJ software (Schneider et al., 2012) .
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