Immunoprecipitation Analyses of Estrogen Receptor α Phosphorylated at Serine 216 in the Mouse Liver.

Estrogen receptor α (ERα) conserves a phosphorylation motif at Serine 216. This site constitutes a protein kinase C phosphorylation motif located within the DNA binding domain (DBD) of ERα. The liver plays a critical role in the regulation of metabolism of various xenobiotics, fatty acids, and cholesterol or endogenous compounds. Moreover, numerous metabolizing enzymes are mainly expressed in the liver. In this chapter, we describe several practical experimental procedures confirming that mouse ERα is phosphorylated at serine 216 in livers upon phenobarbital (PB) treatment. Also, this phosphorylation regulates the expression of estrogen sulfotransferase gene (SULT1E1) which has an important role to sulfate and inactivate estrogen. In response to PB, the conserved motif within the DBD activates the Sult1e1 gene. When this motif was mutated, the activation of Sult1e1 was suppressed significantly. This chapter also describes the use of a phospho-peptide antibody (αP-S216) in the chromatin immunoprecipitation (ChIP) assay, and the co-immunoprecipitation (Co-IP) assay visualized by Western blot analysis.