Comprehensive assessment of estrogen receptor beta antibodies in cancer cell line models and tissue reveals critical limitations in reagent specificity

- ERβ is important in prostate and breast cancer, but its role is controversial.
- ERβ antibodies are problematic, with varying specificity.
- We tested a panel of ERβ antibodies and show the most commonly used is non-specific.
- Two antibodies were validated across multiple experimental approaches.
- Using multiple techniques, we show cell lines used to study ERβ lack its expression.

Estrogen Receptor-β (ERβ) has been implicated in many cancers. In prostate and breast cancer its function is controversial, but genetic studies implicate a role in cancer progression. Much of the confusion around ERβ stems from antibodies that are inadequately validated, yet have become standard tools for deciphering its role. Using an ERβ-inducible cell system we assessed commonly utilized ERβ antibodies and show that one of the most commonly used antibodies, NCL-ER-BETA, is non-specific for ERβ. Other antibodies have limited ERβ specificity or are only specific in one experimental modality. ERβ is commonly studied in MCF-7 (breast) and LNCaP (prostate) cancer cell lines, but we found no ERβ expression in either, using validated antibodies and independent mass spectrometry-based approaches. Our findings question conclusions made about ERβ using the NCL-ER-BETA antibody, or LNCaP and MCF-7 cell lines. We describe robust reagents, which detect ERβ across multiple experimental approaches and in clinical samples.