Multiple ERβ antisera label in ERβ knockout and null mouse tissues

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Abstract

In the process of characterizing a custom-made affinity-purified antiserum for estrogen receptor beta (ERβ), ck5912, we used a number of common tests for specificity of ck5912 along with that of 8 commercially available ERβ antisera: Affinity Bioreagents PA1-310B, Invitrogen D7N, Upstate 06-629, Santa Cruz H150, Y19, L20, 1531, and Abcam 9.88. We tested their recognition of recombinant ERβ (rERβ) versus rERα, ERβ versus ERα transfected into cell lines, as well as labeling in wildtype (WT) versus estrogen receptor beta knockout (βERKO) and null (ERβSTL−/L−) mouse ovary, hypothalamus, and hippocampus. To our surprise, we found that while most of these antisera passed some tests, giving the initial impression of specificity, western blot analysis showed that all of them recognized apparently identical protein bands in WT, βERKO and ERβSTL−/L− tissues. We share these results with the goal of helping other researchers avoid pitfalls in interpretation that could come from use of these ERβ antisera.

Introduction

The discovery of estrogen receptor β (ERβ; Kuiper et al., 1996) opened a new door to understanding physiological actions of estradiol. Neuroscientists who study hormone effects in the brain are particularly interested in ERβ because pharmacological and knockout studies point to ERβ as being important in learning and memory (Liu et al., 2008), anxiety (Imwalle et al., 2005, Tomihara et al., 2009), and aggression (Ogawa et al., 1999).

First cloned in rat and subsequently in human and mouse, the ERβ gene contains eight exons and shares a high degree of sequence homology with estrogen receptor alpha (ERα) in the DNA and ligand binding domains (Kuiper et al., 1996, Mosselman et al., 1996, Tremblay et al., 1997). Additionally, the ERβ gene undergoes alternative splicing leading to the expression of several isoforms. One splice variant, ERβ2, contains a 54 bp insert leading to an additional 18 amino acids; other splice variants, called delta variants, lack entire exons (Chu and Fuller, 1997, Lu et al., 1998). Like ERα, ERβ is traditionally thought of as a transcription regulator. However, estradiol also has many rapid effects on neurons that likely involve ER signaling outside of the nucleus and some pharmacological evidence implicates ERβ in these rapid, extranuclear effects (Zhao and Brinton, 2007, Kramár et al., 2009).

Understanding the function of ERβ requires knowing where it is located and what proteins it interacts with, which in turn, requires reliable and specific antibodies. Early studies with ERβ antisera showed some agreement but also some discrepancies between localization of ERβ immunoreactivity (Li et al., 1997, Shughrue and Merchenthaler, 2001) and mRNA (Shughrue et al., 1997), raising concerns about ERβ antisera (Warner et al., 2003, Shughrue and Merchenthaler, 2001). Then, in 2001, the Z8P ERβ antiserum showed consensus between ERβ mRNA and protein expression in many brain areas, including areas that previously were controversial (Shughrue and Merchenthaler, 2001). Unfortunately, however, Z8P is no longer available.

Our lab is particularly interested in ERβ function in the hippocampus, including its colocalization with other proteins. To facilitate studies of ERβ, we produced two ERβ antisera raised in chicken for use in conjunction with other commercially available antisera. The more promising of these was ck5912. In the process of characterizing ck5912, we performed a number of commonly used tests for its specificity, along with the specificity of 8 commercially available ERβ antisera. To our surprise, we found that while some of these antisera passed many tests, some did not. Most significantly, we found that all the ERβ antisera we tested detected immunoreactivity in tissues from two independently generated strains of ERβ knockout mice, the βERKO mouse (Krege et al., 1998) and Chambon's recently generated ERβ null mouse (ERβSTL−/L−; Antal et al., 2008). We share these findings with the goal of helping other researchers avoid pitfalls in interpretation that could arise from the use of these ERβ antisera.

Section snippets

Animals

All animal procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Northwestern University Institutional Animal Care and Use Committee. βERKO and C57/BL6J breeder mice were purchased from Jackson labs. Mice used for experiments were obtained by in-house breeding with genotype confirmation by Transnetyx. Tissues from ERβSTL−/L− and wildtype mice were a kind gift from Dr. Shaila Mani (Baylor College of

Results

Our initial test of ERβ antisera was to confirm specificity for rERβ versus rERα using western blot. Gels were loaded with 0.1 μg rERβ and rERα, and then probed with one of 9 ERβ antisera: PA1-310B, ck5912, D7N, H150, 9.88, 06-629, 1531, L20, or Y19 (Fig. 1A–I). All antisera detected a band of ∼50 kDa for rERβ. D7N, 06-629, and 1531 also recognized a band >76 kDa (Fig. 1C, F and G), which could correspond to a cluster of rERβ; however this was not seen with the other antisera. Additionally, both

Discussion

We initially set out to characterize a new affinity-purified anti-ERβ antiserum produced in chicken, ck5912. We included 8 commercially available ERβ antisera as controls: Affinity Bioreagents PA1-310B, Invitrogen D7N, Upstate 06-629, Santa Cruz H150, L20, Y19, and 1531, and Abcam 9.88. We first tested recognition of recombinant ERα and ERβ, then detection of ERα and ERβ in transfected cell lines, followed by the most important test, labeling in tissues from WT versus βERKO and ERβSTL−/L− mice.

Acknowledgements

This work was supported by the Northwestern University Institute for Women's Health Research, National Institute of Neurological Disorders and Stroke Grant R01 NS037324, National Institute of Mental Health Grant T32 MH067564.

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