Figure 1

Evidence for the specificities of select commercially available HOXA4 antibodies. Immunoblot analysis was performed as in [3] with lysates from one human acute monocytic leukemia (THP-1) and five ovarian cancer (OVCAR-8, OVCAR-3, SKOV-3, A2780 and CaOV-3) cell lines. Red arrowheads indicate HOXA4 (~37-39 kDa), black arrowheads indicate the ~30-33 kDa non-specific band associated with the Abcam antibody, and white arrowheads indicate select non-specific bands of equal/greater intensity. (A) SKOV-3 cells lack HOXA4 whereas OVCAR-8 and OVCAR-3 cells express high levels of HOXA4 [2, 3]. Transient knockdown was performed as in [3] with 20 nM HOXA4-targeting siRNA (siHOXA4; ON-TARGETplus SMARTpool; Dharmacon), control siRNA (siControl; ON-TARGETplus Non-Targeting Pool) or transfection reagent alone (iMAX; Lipofectamine RNAiMAX). Immunoblot analysis with the Abcam HOXA4 antibody characterized by us [2, 3] shows that the ~30-33 kDa non-specific band detected by Lillvis et al. [1] is insensitive to HOXA4 siRNA and is expressed by HOXA4-negative SKOV-3 cells. (B) A2780 cells lack HOXA4 whereas CaOV-3 cells express low levels of HOXA4 [2, 3]. CaOV-3 cells were transfected as in [3] with control vector (CaOV-3-Vector) or vector encoding full-length HOXA4 (CaOV-3-HOXA4). Immunoblot analysis with the Abcam antibody and two other commercially available HOXA4 antibodies shows that the ~37-39 kDa HOXA4 band detects exogenously expressed HOXA4 and is undetectable in HOXA4-negative A2780 cells. (C) Formaldehyde-fixed SKOV-3 cells were permeabilized, blocked and incubated overnight with anti-HOXA4 (1:3200). Secondary antibody was applied, cell nuclei were stained with Hoechst 33258, and coverslips were examined by epifluorescence microscopy. Although HOXA4 mRNA [2, 3] and protein (A) are undetectable in SKOV-3 cells, these cells have strong expression of the ~30-33 kDa non-specific band ((A) and [2]). Note that immunofluorescent staining of HOXA4-negative SKOV-3 cells with the Abcam antibody yields strong perinuclear staining which cannot be attributed to HOXA4.