Example control sample curve. The phosphorylation state of PDHA1 can vary by treatment but also by cell culture conditions such as media supplements, nutrients and also cell density.
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 S293. Conversely NaF treatment, to inhibit cellular serine phosphatases, increased the phosphorylation level of S293.
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 S293 which is increased by kinase treatment. Conversely phosphatase treatment was able to significantly reduce the phospho S293 signal from the endogenous levels.