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Tools for Mitochondrial Metabolism
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 Dysregulation of the pyruvate dehydogenase (PDH) complex (PDC) in skeletal muscle has been implicated in type 2 diabetes and various mitochondrial diseases. The activity of the PDC complex is mainly regulated by the phosphorylation state of Ser293 on the E1a subunit. Phosphorylation inactivates the complex, while removal of the phosphate activates the complex . We have developed a novel and specific antibody to measure phosphorylation of PDH-E1a at Ser293. The antibody detects only the phosphorylated form of PDH-E1a in rat brain, heart, and liver mitochondrial homogenates, human tissue culture whole cell lysates, as well as in intact cells using fluorescent immunocytochemistry. COS7 cells treated with the anti cancer drug dichloroacetate (DCA), a w ell characterized mitochondrial pyruvate dehydrogenase kinase (PDK) inhibitor, display a complete loss of mitochondrial specific fluorescent phospho PDH-E1a staining (see figure).This unique antibody will allow the facile measurement of flux through the glycolytic pathway by monitoring PDH phosphorylation as a readout of activity. The reagent should prove a useful tool in models of diabetes and neuronal pathogenesis for assessing the signals required for coordinating mitochondrial metabolic responses to a large array of stimuli including glucose levels, reactive oxygen species, apoptotic responses, and extracellular growth signals.
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PhosphoDetect™ Anti-PDH-E1a (pSer293) Rabbit pAb (Cat. No. AP1062) | |
Detection of PDH-E1a, phosphorylated at Ser293 : COS7 cells incubated in the absence (top 3 panels) and presence (middle 3 and bottom 3 panels) of dichloroacetate (DCA; 5 mM for 2 h). Phosphorylated PDH (green), mitochondria (red), and nuclei (blue). Data courtesy of Sandra Wiley and Matthew Rardin, University of California San Diego. |
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Detection of human PDH-E1a, phosphorylated at Ser293, by immunoblotting. Samples: Whole cell lysate (20 mg) from HEK293 cells incubated in complete growth medium (lane 1) and incubated in complete growth medium and treated with (DCA, 5 mM for 4 h). Primary antibody: PhosphoDetect™ Anti PDH-E1a (pSer293) Rabbit pAb (Cat. No. AP1062) (0.25 mg/ml). Detection: chemiluminescence.
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