( C) Effects of SRC-1 on the transactivation activities of ER-β isoforms. Binding data were calculated and analyzed with GraphPad Prism 4.0 software to determine the B max and K d of each isoform. Four hundred micrograms of total yeast lysate expressing ER-β isoforms was applied to each binding reaction as described in Materials and Methods. ( B) Tabulated results of in vitro estrogen receptor binding assay. Lanes 1 and 2, 1 and 4, and 1 and 5 represent the samples overexpressing ER-β1 and -β2, ER-β1 and -β4, and ER-β1 and -β5, respectively. Coexpression of ER-β isoforms with ER-β1 was also performed in both cell line and yeast.
The size of the ER-β isoforms was consistent with the predicted molecular size, ranging from 53 to 59 kDa. Samples expressing ER-β1, -β2, -β4, and -β5 were labeled as 1, 2, 4, and 5, respectively.
Mock-transfected cells or an untransformed yeast strain were set up as a control experiment (CTL). An equal amount (50 μg) of protein was loaded to each lane. N-terminal-specific polyclonal ER-β antibody (H150 from Santa Cruz Biotechnology), which would recognize all ER-β isoforms, was used in this study. ( A) Western blot analysis of ER-β isoforms overexpressed in HEK293 cells and yeast.
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