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. 2018 Sep 26;293(47):18387–18399. doi: 10.1074/jbc.RA118.004653

Figure 5.

Figure 5.

The UBE3A G20V and C21Y AS mutants act as repressors of estrogen receptor signaling. HEK293T cells were transfected with an ER-responsive firefly luciferase reporter construct and a construct encoding Renilla luciferase to adjust for transfection efficiency either in the absence or presence of an expression construct for ERα. To determine the effect of different UBE3A variants on ER signaling, increasing amounts of the different HA-tagged UBE3A variants were expressed. The effect of ERα on the reporter construct was measured using a Dual-Luciferase assay. The values of firefly luciferase were normalized to those of Renilla luciferase and then expressed as relative values to induced (only ERα-expressing) cells. The depicted graph shows data points collected in six independent experiments. Bars represent means and 95% confidence intervals. Open gray circles represent 1.4 μg of transfected UBE3A plasmid; closed black circles represent 2.4 μg of transfected UBE3A plasmid. A Western blot showing expression levels of transfected HA-UBE3A variants of one representative experiment is shown below; loaded lysate amounts have been adjusted for Renilla luciferase activity. UBE3A was detected using anti-HA antibody.