Ab191178

Cell lysates were extracted in 0.1 M NaCl, 0.01 M tris–HCl (pH 7.6), 1 mM EDTA (pH 8.0), 1 mg/ml aprotinin, and 100 mg/ml PMSF; protein concentrations were determined by a Bio-Rad protein assay. Protein (50 μg) was boiled at 95 °C in sodium dodecyl sulphate (SDS) sample buffer for 5 min, electrophoresed on 10% or 12% SDS-PAGE gels, and transferred to polyvinyldifluoridine membranes. Then, blots were incubated overnight at 4 °C with anti-HOXA9 (Abcam, Cambridge, UK, #ab191178; 1:1000), anti-RUNX2 (Abcam, Cambridge, UK, #ab23981; 1:1000), or anti-β-actin (Bethyl, Montgomery, Texas, USA, #A300-491A; 1:8000) antibodies. Membranes incubated with anti-goat or anti-rabbit secondary antibody (Santa Cruz Biotechnology; 1:5000) at room temperature for 60 min. Protein band signals were visualized using an ECF western blotting kit (Amersham Biosciences, Piscataway, NJ, USA) and detected with an LAS3000 luminescent image analyzer (Fuji Photo Film).

Cells were lysed in the RIPA lysis buffer that included a protease inhibitor cocktail on ice. An equivalent amount of total protein was separated from various samples using SDS-PAGE gels at 100 V for 1.5 h and then transferred onto 0.22-μm polyvinylidene difluoride membranes at 280 mA for 1.5 h. The membranes were blocked with 5% non-fat milk for 1 h and then incubated overnight at 4°C with primary antibodies. The primary antibodies were: anti-HOXA9 (ab191178, 1:500), anti-CyclinD1 (ab134175, 1:5000), anti-c-Myc (ab185656, 1:1000), and anti-β-catenin (ab16051, 1:1000; all from Abcam, San Francisco, USA). Membranes were washed and then treated with goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) for 1 h at 37°C. Protein bands were visualized using chemiluminescence HRP substrate, where β-actin served as an internal control. Blot density was then quantified using Image J software.

Expression of HOXA4, HOXA9, and HOXB4 in mesothelioma and normal mesothelium tissue was investigated using 3 μm-thick, formalin fixed, paraffin embedded tissue array sections (MS081, US Biomax, Rockville, MD, USA). Immunohistochemical analysis was performed using a monoclonal rabbit anti-HOXB4 antibody (ab676093, 1:100 dilution, Abcam, Cambridge, UK), a polyclonal rabbit anti-HOXA4 antibody (ab131049, 1:500 dilution, Abcam, Cambridge, UK), and a polyclonal rabbit anti-HOXA9 antibody (ab191178, 1:75 dilution, Abcam, Cambridge, UK). The ABC detection method with peroxidase block (DakoCytomation) was used for all of these primary antibodies. Antigen retrieval was performed using pH 9.0 Tris/EDTA buffer (DakoCytomation) and heating in a microwave for 23 min.