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.