HOXA4 protein levels and localization in the aorta and in human abdominal aortic aneurysms

We read with interest the study of Lillvis et al. [1] regarding the expression of HOXA4 in the aorta and its potential role in abdominal aortic aneurysms. The authors used microarray analysis validated by reverse transcription quantitative real-time PCR to provide strong evidence that HOXA4 mRNA levels are reduced in human abdominal aortic aneurysms relative to control human abdominal aorta. However, we have significant concerns about the subsequent data regarding HOXA4 protein levels. For their studies Lillvis et al. used a commercially available rabbit polyclonal HOXA4 antibody (ab26097; Abcam, Cambridge, MA) that was previously characterized extensively by us [2,3]. While they were kind enough to reference our studies, they state that HOXA4 was detected as a single band at ~33 kDa and evidence for this is presented in their Additional file three, Figure S1A. However, in both of our previous studies we state that the size of HOXA4 is ~37-39 kDa and our first study [2] demonstrated that the band at ~30-33 kDa is a non-specific band.The ~30-33 kDa non-specific band is by far the most intense band and appears as a single band at low exposures irrespective of blotting conditions, as was observed by Lillvis et al. in Additional file three, Figure S1A. This was demonstrated previously by us [2] and we now provide additional evidence of this in Figures 1A and 1B. Strong expression of the ~30-33 kDa non-specific band is observed in the five human ovarian cancer cell lines shown in Figures 1A and 1B regardless of the fact that HOXA4 mRNA is undetectable in both SKOV-3 and A2780 cells [2,3]. More importantly, the ~30-33 kDa non-specific band is insensitive to small interfering RNA (siRNA) targeting HOXA4 ([2] and Figure 1A) and forced expression of full-length HOXA4 (Figure 1B). Likewise, Figure 1A shows strong, siRNA-insensitive expression of the ~30-33 kDa non-specific band in the acute monocytic leukemia cell line used by Lillvis et al. (THP-1 cells, designated as MP1 ce