Detection of PDH-E1a, phosphorylated at Ser²⁹³ by immunocytochemistry. Samples: COS7 cells incubated in the absence (top 3 panels) and presence (middle 3 and bottom 3 panels) dichloroacetate (DCA; 5 mM for 2 h). All samples were incubated with MitoTracker^® Red stain (100 nM for 20 min; to visualize mitochondria) and fixed in 3.7% formaldehyde, followed by methanol (3 min at -20°C), and permeabilization in PBS, 6% BSA, 0.3% Triton™ X-100 detergent, 0.1% Tween^® 20 detergent. Primary antibodies: PhosphoDetect™ Anti-PDH-E1a (pSer²⁹³) Rabbit pAb (Cat. No. AP1062) (0.5 µg/ml, green fluorescence, top 3 and middle 3 panels) or anti-PDH-E1a antibody (green fluorescence, bottom 3 panels). Detection: fluorescence (Alexa Fluor^® 488 secondary antibody) with DAPI counterstain (300 nM, 1 min, blue fluorescence). Data courtesy of Sandra Wiley and Matthew Rardin, University of California San Diego.

Detection of human PDH-E1a, phosphorylated at Ser²⁹³, by immunoblotting. Samples: Whole cell lysate (20 µg) from HEK293 cells incubated in complete growth medium (lane 1) and incubated in complete growth medium and treated with dichloroacetate (DCA, 5 mM for 4 h). Primary antibody: PhosphoDetect™ Anti PDH-E1a (pSer²⁹³) Rabbit pAb (Cat. No. AP1062) (0.25 µg/ml). Detection: chemiluminescence. Data courtesy of Sandra Wiley and Matthew Rardin, University of California San Diego.