Skip to main content
. 2015 Dec 9;35(49):16077–16093. doi: 10.1523/JNEUROSCI.1890-15.2015

Figure 3.

Figure 3.

Analysis of functionality and in vivo expression of viral vectors. A, Plasmids expressing cmyc-tagged ERα, cmyc-tagged ERβ, or GFP were cotransfected with an ERE-SEAP reporter plasmid in HEK293T cells with the addition of 1 or 10 nm EB treatment or vehicle. SEAP reporter levels were measured 24 h after application of treatments and compared with control groups. These results confirm functionality of the encoded ERs in vitro. Asterisks indicate a significant (p < 0.05) increase in SEAP expression relative to control. B, Western analysis of HEK293T cells treated with AAV-ERα, AAV-ERβ, or the GFP control virus. Top, For cells treated with AAV-ERα, antibodies for ERα (Ab17) and cmyc-tag (A21281) detected the 67 kDa full-length protein for ERα. Bottom, For cells treated with AAV-ERβ, antibodies for ERβ (H-150) and cmyc-tag (A21281) detected a band at ∼56 kDa. Note that HEK293T cells treated with the GFP virus exhibit a band at ∼56 kDa, which is increased in cultures treated with AAV-ERβ-cmyc. C, D, Immunofluorescent staining for the cmyc-tag, verified hippocampal expression of ERα and ERβ 4 weeks after injection of ER-encoding vectors. AAV-GFP was mixed with the ER virus in a 1:4 ratio to visualize distribution of expression vectors.