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. Author manuscript; available in PMC: 2014 Dec 10.
Published in final edited form as: Endocrinology. 2007 Jul 19;148(10):4774–4785. doi: 10.1210/en.2007-0537

Fig. 3. Regulation of Bip in the uteri of wild-type and ERα(−/−) mice by kepone.

Fig. 3

A, Dose response studies. Adult ovariectomized wild-type mice were injected (sc) with kepone at 7.5, 15, 30 mg/kg, and killed at 24 h. Uterine tissue extracts (30 μg protein) were analyzed by Western blotting for Bip, ERα and Actin. B, Temporal studies. Adult ovariectomized wild-type mice were given a single injection (sc) of kepone at 7.5 mg/kg and analyzed at indicated times. Additionally, mice were injected with multiple (3×) injections of kepone at 7.5 mg/kg and killed 24 h after the last injection [24(M)]. Uterine tissue extracts (30 μg protein) were analyzed by Western blotting for Bip, ERα and Actin. C, (i) Western blot analysis. Regulation of Bip by kepone in the uteri of wild-type and ERα(−/−) mice. Adult ovariectomized wild-type and ERα(−/−) mice were given injections (sc) of oil, kepone (15 mg/kg), ICI (20 mg/kg) or the same dose of ICI 30 min prior to kepone, and mice were killed at 24 h. Uterine tissue extracts (20 μg protein) were analyzed by Western blotting for Bip and Actin. C, (ii) Northern blot analysis. ICI effectively blocks uterine LF regulation by E2 in the uteri of wild-type mice. Adult ovariectomized wild-type mice were given injections (sc) of oil, E2 (4 μg/mouse), ICI (20 mg/kg) or the same dose of ICI 30 min prior to E2, and mice were killed at 24 h. In general, 3-5 mice were used for each group in above analyses (A-C). These experiments were repeated three times with independent samples. In the bar plot, the abundance of each protein expression was quantitated by analysis of band intensities using densitometric scanning and was corrected against Actin. *Values are statistically different (p<0.05, student’s t test) from the corresponding control group. **Values are statistically different (p<0.05, student’s t test) from the E2-treated group. D, Analysis of protein-protein interaction. Uterine tissue extracts for the indicated groups were subjected to immunoprecipitation using Bip antibody, followed by Western blotting using specific antibodies to Bip (upper panel) or ERα (lower panel). The intense bands represent heavy chain subunit of IgG (this also serves as an internal loading control). In our control experiments, immunoprecipitation using normal goat serum did not detect any specific bands for Bip or ERα by Western blotting (data not shown). These experiments were repeated two times with similar results.