10 μg of protein lysates were resolved by reducing 12% SDS-PAGE and transferred to nitrocellulose membranes Hybond-C (Amersham). After electrophoresis, protein check details transfer was verified by Ponceau staining. The nitrocellulose membranes were probed with antibodies anti-SIAH-1

and anti-Kid/KIF22 (both diluted 1:1000) followed by horseradish peroxidase-coupled secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) anti-chicken IgG (diluted 1:2000) or anti-rabbit IgG (diluted 1:2500) and detected using a chemiluminescence-based detection system (ECL, Amersham). Immunofluorescence staining Paraffined tissue array slides containing 20 normal and 19 matched malignant human tumor tissues, or 25 cancerous and 4 normal breast

human tissues were obtained from Imgenex (Clinisciences, France), and processed as per manufacturer recommendations. Breast tumors and normal surrounding tissues from the same patients were obtained by sectioning frozen tissues. The slides were fixed in 2% paraformaldehyde (PFA) PD0332991 nmr for 10 min at room temperature (RT) and washed in PBS six times. Nonspecific protein binding was blocked by incubation in a PBS solution containing 3% BSA, 0.1% saponin for 2 h at RT. Slides were then incubated overnight with primary antibody diluted in 0.3% BSA, 0.1% saponin in PBS at

4°C. After six washes with PBS, staining was revealed using a Rhodamine Red-X-conjugated secondary antibody for SIAH-1 and FITC-conjugated secondary antibody for Kid/KIF22 (Jackson Labs). Slides were subsequently analysed Tryptophan synthase using a Zeiss epifluorescence microscope equipped with a cooled three-charged coupled device (3CCD) camera (Lhesa, France), triple band pass filter and a high numerical aperture lens (40 × 1.3 NA and 100 × 1.3 NA). Results Analysis of SIAH-1 in human tissues and cell lines extracts The expression of SIAH-1 in a variety of human tissues and human derived cell lines was explored by western blotting using SIAH-1 anti-sera (previously described [17]), (Figure 1). Two major bands with an apparent MW of ~35 kDa and ~70 kDa were detected in human brain, heart, small intestine, Kidney and pancreas extracts. In contrast, no bands were evident in human lung, testis and spleen extracts (Figure 1a). The smooth muscle extract showed only the minor band of ~35 kDa. In addition, an extra band of ~52 kDa was detected in brain, liver and pancreas extracts. Besides the two principal bands, additional bands of higher molecular weight showing a ladder pattern were detected in small intestine and pancreas extracts. This profile is characteristic of polyubiquitinated proteins.