2 Western blotting analysis showing representative breast cancer sera recognizing p90/CIP2A recombinant protein
2 Western blotting analysis showing representative breast cancer sera recognizing p90/CIP2A recombinant protein. individuals (2.3 %). The frequency of p90/CIP2A expression in breast cancer tissues was significantly higher than that in adjacent normal tissues ( 0.01). Our preliminary results suggest that autoantibodies against p90/CIP2A may be a useful serum biomarker for early stage breast cancer screening and diagnosis. for 10 min. The supernatant was used for immunofluorescence assay. Immunohistochemistry (IHC) with tissue array slides Breast cancer tissue array slides with normal tissue controls (46 cases/92 cores, including clinical stages and pathology grades) were purchased (US Biomax, Inc., Rockville, MD) and used to detect the expression of the p90/CIP2A protein. The slides were deparaffinized with xylene and dehydrated with ethanol of different strengths. Antigen retrieval was performed by microwave-heating methods in Trilogy? pretreatment solution for 20 min and cooled down naturally for about 1 h. Three percent H2O2 and 10 %10 % fetal bovine serum blocking solution were used to prevent nonspecific binding of antibodies for 20 min separately. The sections were incubated with monoclonal anti-p90/CIP2A antibody (1:500 dilution) overnight at 4 C. HRP Detection System (HRP streptavidin label and polyvalent biotinylated link) and DAB Substrate Kit were used as detecting reagents. After counterstaining with hematoxylin, the sections were dehydrated and mounted. Finally, the slides were observed by a microscope (Leica, DM1000). All IHC results were read blindly by two impartial researchers. A four-level scoring system (?, unfavorable; +, low expression level; ++, moderate expression level; +++, high expression level) was used to evaluate the staining intensity. Statistical analysis Statistical analysis was performed using SPSS 13.0. Data were analyzed with 2 test and represented as the mean3 SD from ELISA. The results were considered to indicate a statistically significant difference when values were 0.05 or 0.01. Results Frequency and titer of anti-p90/CIP2A autoantibody in sera from patients with breast cancer Serum levels of anti-p90/CIP2A autoantibodies were determined by ELISA as described in the section of Materials and methods. In total, 168 sera from PP121 patients with breast cancer and 88 sera from normal human individuals were used in this study. As Pou5f1 shown in Table 1, the prevalence of autoantibody against p90/CIP2A was 19.1 % (32/168) in breast cancer, which was significantly higher than that in NHS (2.3 %, 2/88) ( 0.01). Titer of anti-p90/CIP2A antibodies in human sera is shown in Fig. 1. The average titer of autoantibody against p90/CIP2A in breast cancer sera was higher than that in NHS ( 0.01). The ELISA PP121 results were also confirmed by western blot analysis. Figure 2 shows that representative breast cancer serum with positive reaction to p90/CIP2A in ELISA also has strong reactivity in western blotting compared to normal serum. Open in a PP121 separate window Fig. 1 Titer of autoantibody against p90/CIP2A in human sera by ELISA. The range of antibody titers to p90/CIP2A was expressed as optical density (OD) obtained from ELISA. The mean+3 SD of NHS are shown in relationship to all serum samples. Titer of anti-p90/CIP2A in breast cancer is much higher than that in NHS ( 0.01) Open in a separate window Fig. 2 Western blotting analysis showing representative breast cancer sera recognizing p90/CIP2A recombinant protein. The monoclonal anti-p90/CIP2A antibody was used as positive control; and value relative to NHS: ** 0.01 Immunofluorescence staining pattern of p90/CIP2A in HEp-2 cells To further confirm the reactivity of autoantibody against p90/CIP2A in breast cancer sera and the intracellular localization of p90/CIP2A, HEp-2 cell slides were used in indirect immunofluorescence assay to detect breast cancer sera with anti-p90/CIP2A positive in ELISA. As shown in Fig. 3, a representative anti-p90/CIP2A positive breast cancer serum had both cytoplasmic and perinuclear staining pattern, while the monoclonal anti-p90/CIP2A antibody with more intense staining in perinuclear regions. The fluorescent staining was significantly reduced when the same breast cancer serum was pre-absorbed with recombinant p90/CIP2A protein. Open in a separate window Fig. 3 Representative immunofluorescence staining pattern of anti-p90/CIP2A antibody positive breast cancer serum. a A normal human serum (NHS) was used as unfavorable control; b monoclonal anti-p90/CIP2A antibody that exhibited a perinuclear immunofluorescence staining pattern was used as positive control; c a representative anti-p90/CIP2A antibody positive breast cancer serum exhibited both cytoplasmic and perinuclear immunofluorescence staining pattern; d the same breast cancer serum that was used in c was post-absorbed with recombinant p90/CIP2A protein. The fluorescent signal was remarkably decreased Expression of p90/CIP2A in breast cancer tissues and adjacent normal breast tissues by immunohistochemistry In the present study, the expression of p90/CIP2A in breast cancer tissues and adjacent normal breast tissues was examined by immunohistochemistry with tissue array slides. Tissue array slides including 46 breast cancer tissue specimens and 46 adjacent normal breast tissue specimens were commercially available for this study. The monoclonal.