Fig. 1.

Validation of ERβ antibodies using doxycycline-inducible MDA-MB-231-ERβ cells. (A) MDA-MB-231-ERβ cells were treated with doxycycline to induce ERβ expression. Untreated cells provided an ERβ-negative control. Messenger RNA was extracted for qRT-PCR and protein for Western blotting. MDA-MB-231-ERβ+ and MDA-MB-231-ERβ– cells were crosslinked and immunoprecipitated with antibody for RIME. (B) qRT-PCR confirmed 100-fold induction of ERβ mRNA in MDA-MB-231-ERβ+ cells versus untreated MDA-MB-231-ERβ– cells. Data are mean ± S.D. of technical triplicate experiments. (C) Western blots of MDA-MB-231-ERβ+ and MDA-MB-231-ERβ– cells with the 8 antibodies undergoing assessment. The MC10, CWK-F12, Abcam 288[14C8] and sc8974 antibodies detected bands of 59 kDa, with differential signal in the ERβ+ versus ERβ– conditions, indicating specificity to ERβ. GeneTex 70182 detected ERβ, although there was non-specific signal at 65 kDa. Millipore 06-629 appears to detect ERβ, although there is also a 59 kDa band in the ERβ– condition. Review of the RIME data suggests this may be cross-reactivity with LACTB. NCL-ER-BETA, the most commonly used ERβ antibody, gives bands of the correct size for ERβ, but there is no difference between ERβ– and ERβ+ conditions, confirming that this antibody is not specific to ERβ.