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. Author manuscript; available in PMC: 2011 Oct 15.

Published in final edited form as: Arch Biochem Biophys. 2010 Jul 29;502(2):121–129. doi: 10.1016/j.abb.2010.07.022

Fig. 4 — Effect of Ainp2 on the E2-activated, ER-mediated luciferase activity in Arnt knockdown MCF-7 cells. A. RT/real-time qPCR showing a decrease of the endogenous Arnt message after transfection of 160 nM Arnt siRNA. Error bar represents the standard deviation of the means (n = 2, means ± SD) of the amount of Arnt normalized to the corresponding 18S. B. Chemiluminescent Western analysis showing the reduced expression of the Arnt protein after gene silencing. 10 μg of nuclear extracts was separated on a 10% SDS-PAGE gel, followed by the Western blot using the anti-Arnt antibodies (sc-8077, Santa Cruz, 1:500). The arrow indicates the Arnt protein. C. Luciferase assay showing that the Ainp2 effect in the E2-activated ER signaling was Arnt-dependent. Transient transfection was performed using Lipofectamine 2000. All conditions contained 10 nM E2 and the same amount of transfected plasmids of different combinations (pCMV-Tag4 or pCMV-Tag4-Ainp2). The normalized luciferase activity of the empty vector (pCMV-Tag4) was arbitrarily set as 1 and used to calculate the relative luciferase activity. All conditions were performed in six replicates (n =6) except for the control (n = 12). ** p ≤ 0.005, † p > 0.05 (not significant).

Fig. 4 — Effect of Ainp2 on the E2-activated, ER-mediated luciferase activity in Arnt knockdown MCF-7 cells. A. RT/real-time qPCR showing a decrease of the endogenous Arnt message after transfection of 160 nM Arnt siRNA. Error bar represents the standard deviation of the means (n = 2, means ± SD) of the amount of Arnt normalized to the corresponding 18S. B. Chemiluminescent Western analysis showing the reduced expression of the Arnt protein after gene silencing. 10 μg of nuclear extracts was separated on a 10% SDS-PAGE gel, followed by the Western blot using the anti-Arnt antibodies (sc-8077, Santa Cruz, 1:500). The arrow indicates the Arnt protein. C. Luciferase assay showing that the Ainp2 effect in the E2-activated ER signaling was Arnt-dependent. Transient transfection was performed using Lipofectamine 2000. All conditions contained 10 nM E2 and the same amount of transfected plasmids of different combinations (pCMV-Tag4 or pCMV-Tag4-Ainp2). The normalized luciferase activity of the empty vector (pCMV-Tag4) was arbitrarily set as 1 and used to calculate the relative luciferase activity. All conditions were performed in six replicates (n =6) except for the control (n = 12). ** p ≤ 0.005, † p > 0.05 (not significant).