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 did not reduce the level of phospho S300 confirming that there is little endogenous phospho S300. Conversely NaF treatment, to inhibit cellular serine phosphatases, did increase the phosphorylation level of S300.
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 did not show a significant endogenous phosphorylation signal at S300 this could be increased by kinase treatment. Phosphatase treatement had no effect on the endogenous (low) level of phospho S300 signal.