Attenuated HRH4 expression in CRCs. (A) Representative blots of HRH4 expression in normal mucosa and colorectal tumor tissues. Sample 1, 2: Dukes A; sample3, 4: Dukes B; sample5, 6: Dukes C; sample7, 8: Dukes D. GAPDH was used as a stable endogenous control. (B) Analysis of HRH4 protein level in CRC samples. Quantitative image of blot were calculated using TotalLab TL100 software, and quantities of GAPDH were used to normalize the HRH4 expression data. The expression level of HRH4 in each sample was calculated as the ratio between CRC tissues and matched adjacent normal tissue. Each data was obtained from two independent results of immunoblottings. (C) Real-time PCR assay was carried out as described under Materials and Methods section, boxplots of relative HRH4 mRNA(HRH4/GAPDH) measured with real-time PCR analysis showing median; box: 25th -75th percentile; bars: largest and smallest values within 1.5 box lengths; little circles: outliers. The results were obtained from 3 reactions in each sample. (D) Representative immunofluorescent microscope analysis of paired samples of CRC tissue and adjacent normal tissue using anti human HRH4 monoclonal antibody (red). Nuclei were stain with DAPI (blue). Sample1: rectum tissue; sample2: colon tissue, white arrow points to the positive control of HRH4 expression on blood vessel epithelium. (E) Representative immunohistochemical staining of paired samples of CRC tissue and adjacent normal tissue using anti human HRH4 monoclonal antibody. Sample1: rectum; sample2: colon tissue. Black arrow points to the positive expression of HRH4 expression on enterocytes.