Development of monitoring method for the alteration of phosphorylation status in proteasome subunits using Phos-tag electrophoresis

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The phosphorylation of proteasome subunits is thought to play an important role in the function of proteasome. However, the relationship between the phosphorylation of proteasome subunits and the regulation of functions has not been examined, and the phosphorylated status of proteasome subunits has not been biochemically characterized in detail. To monitor the alteration of phosphorylation status in proteasome subunits, the phosphorylated forms of each subunit were investigated using Phos-tag electrophoresis followed by immunoblotting. The results indicated that Phos-tag electrophoresis enabled us to separate and detect the phosphorylated forms of two proteasome subunits. Furthermore, Phos-tag electrophoresis indicated that the phosphorylation status in these proteasome subunits was modulated in response to external stimulation with hydrogen peroxide.