Western immunoblot analysis of NUCKS in primary cultures from different biopsies. (A) Equal amounts of total protein (30 μg) were subjected to immunoblot analysis with anti-NUCKS antibody. After stripping, the membrane was reprobed with anti-actin antibody. Actin protein was used as an internal control and in order to verify equal loading of the gel. MDA MB-231 and MCF-7 cell lines were used as a positive control. The most representative cases are illustrated. (B) Graphical presentation of the ratio of NUCKS to actin protein levels corresponding to the samples illustrated in (A), as median values of 2 independent experiments (p = 0.05). The protein levels were calculated from the immunoblot reaction with the Image-Pro Plus software, as described in the Methods section. TC01, primary culture of normal tissue; TC05, primary culture of fibroadenoma; TC11 and TC13, derived from primary cultures from biopsies with benign epithelial proliferations; TC16, TC20, TC27-TC31 derived from primary cultures from grade II breast cancer biopsies; TC32 and TC36 derived from IDC, grade III. NUCKS protein levels resemble its mRNA expression, exhibiting the same variation among the different samples except for IDC grade III. MDA MB-231 and MCF-7 represent cell lines used as a positive control for NUCKS expression. The clinicopathological variables of the samples are summarized in Additional file 1. (C) Median values of the ratio of NUCKS to actin protein levels in the studied groups (p = 0.05).