The PDHA1 bound from undosed HeLa cells was subject to in-well kinase treatment (PDK1&3) or in-well phosphatase treatment (PDP1) according to the supplementary protocol shown below. Untreated cells showed a significant endogenous phosphorylation signal at S232 which is only slightly increased by kinase treatment. Conversely phosphatase treatment was able to significantly reduce the phospho S232 signal from the endogenous levels.
HeLa cells were cultured for 4 hours in media supplemented with DCA (20mM) to specifically inhibit mitochondrial PDH kinases, or NaF (20mM), a general inhibitor of serine/threonine protein phosphatases. The DCA treatment reduced the level of phospho S232. Conversely NaF treatment, to inhibit cellular serine phosphatases, increased the phosphorylation level of S232.
Example control sample curve for HepG2 cell extract. The phosphorylation state of PDHA1 can vary by treatment but also by cell culture conditions such as media supplements, nutrients and also cell density.
Example control sample curve for HeLa cell extract. The phosphorylation state of PDHA1 can vary by treatment but also by cell culture conditions such as media supplements, nutrients and also cell density.