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. 2013 Sep 25;25(1):1–13. doi: 10.1089/hgtb.2013.014

FIG. 6.

FIG. 6.

Reversible tamoxifen-induced ATOH1-ER-DSRED fusion protein expression of the endogenous ATOH1 protein. HEK cells were transiently transfected with the Atoh1-ER-DsRed construct and incubated with different doses of tamoxifen for 72 hr, and cytosolic or nuclear protein was collected and processed for Western blot analysis for ATOH1 expression. Positive control samples were transfected with a cmv.Atoh1 construct consisting of consecutive flag-tag sequences, and negative control samples were transfected with the DsRed-ER construct. (A) Ani-ATOH1 labeling of the cytosolic fraction isolated from HEK cells transfected with either Atoh1-ER-DsRed or cmv.Atoh1 and incubated with 1 μM tamoxifen for 72 hr illustrates the relative expression between the 110 kDa ATOH1-ER-DSRED fusion protein (a), 47 kDa flag-tagged ATOH1 protein (b), and 45 kDa endogenous Atoh1 protein (c). (B) The top row shows that increased concentrations of tamoxifen resulted in decreased anti-FLAG immunolabeling of the ∼110 kDa ATOH1-ER-DSRED fusion protein in the cytosolic fraction, and increased immunolabeling of this fusion protein in the nuclear fraction. Labeling for the 110 kDa fusion protein is absent in the cytosolic fraction of cmv.Atoh1 and DsRed-ER-transfected controls. However, immunolabeling for the 47 kDa flag-tagged ATOH1 protein is present in the cytosolic and nuclear fraction at equivalent levels of cells transfected with the cmv.Atoh1 construct. Middle row: Immunolabeling the cytosolic fraction with anti-ATOH1 shows a 47 kDa band corresponding to the transfected flag-tagged cmv.Atoh1 construct (b), and a 45 kDa signal corresponding to endogenous Atoh1 (c). Since ATOH1 is autoregulatory, cmv.Atoh1-transfected cells exhibit an increased intensity of endogenous 45 kDa immunolabeling over the low level ATOH1 expression seen in DsRed-ER-transfected cells. In cells transfected with the Atoh1-ER-DsRed construct, increased tamoxifen levels corresponded with increased immunolabeling of the 45 kDa endogenous ATOH1 protein, and no labeling to the 47 kDa flag-tagged ATOH1 signal is observed. The letters a, b, and c correspond to the positions denoted in (A). Bottom row: Immunolabeling to cytosolic β-actin is shown for a control for gel loading. (C) The cytosolic and nuclear fractions of HEK cells transfected with either Atoh1-ER-DsRed or cmv.Atoh1 were isolated 0, 24, 72, or 96 hr after 72 hr incubation in 1 mM tamoxifen and compared with untreated samples. In the absence of tamoxifen, there was a relatively large concentration of anti-ATOH1 immunolabeling of the 110 kDa fusion protein in the cytosolic fraction compared with the nuclear fraction. Immediately after tamoxifen washout (0 hr), there was an increase in nuclear anti-ATOH1 immunolabeling for both the 110 kDa fusion protein and the endogenous 45 kDa protein that decreased over time to baseline levels at 72 hr. The decrease in the nuclear immunolabeling to the 110 kDa fusion protein corresponded to an increase in cytoplasmic immunolabeling of the 110 kDa fusion protein over this same time. Control cells transfected with cmv.Atoh1 and incubated for 72 hr in the absence of tamoxifen failed to exhibit labeling to the 110 kDa fusion protein in any condition. The letters a, b, and c correspond to the positions denoted in (A).