The housekeeping gene GAPDH (Takara DR3702) was used as an endogenous control. The relative levels of FHL1 gene expression for each sample were calculated using the 2-ct method. Western-blot Antibodies against FHL1 were purchased from Sigma-Alorich (Sigma-Alorich, Saint Louis, USA; selleck chemical monoclonal mouse, WH0002273M1). Aganglionic and ganglionic colon segments of HSCR samples and colon segments of newborn infants were frozen and lysed in buffer. The protein concentration of each lysate was determined using the bicinchoninic acid (BCA) kit according to the manufacture’s protocol. Total protein (90��g) was applied to each lane on 12% SDS-polyacrylamide gels. After electrophoresis, the polyvinylidene fluoride (PVDF) membranes were washed in Tris-buffered saline containing 0.
1% Tween-20, and then incubated with primary antibody (diluted 1:2000) followed by secondary antibody (diluted 1:2000). Immunostained bands were detected with a ProtoBlot II AP System with a stabilized substrate (Promega, Madison, USA). GAPDH protein was used as internal control. Statistical analysis FHL1 expression values are expressed as mean��SEM. Data were analyzed with Student’s T test. P values less than 0.05 were considered to be statistically significant. Results Immunostaining of FHL1 in HSCR patients The HE and immunostaining of FHL1 in 4 HSCR colons and 4 normal colons were accomplished. Circular muscle layer and longitudinal muscle layer were thickening at different extent in aganglionic and ganglionic segment of HSCR. Compared with normal colon the arrangement of circular muscle layer in aganglionic segment of HSCR was disorganized (Fig.
(Fig.1).1). Immunohistologic study revealed that in the ganglionic segment of HSCR, FHL1 was expressed in the ganglia cells in myenteric, submucosa, circular muscle layer and longitudinal muscle layer. However in the aganglionic segment of HSCR we found expression levels of FHL1 in the circular muscle layer, submucosa, and longitudinal muscle layer (Fig.(Fig.11). Figure 1 Immunol staining of FHL1 in colon. A-C: HE staining in normal colon, ganglionic segment and aganglionic segment of HSCR. D-E: FHL1 staining illustrated that the expression was restricted to the ganglia cells in the myenteric, submucosa, longitudinal muscle … FHL1 gene expression in HSCR patients FHL1 mRNA and protein expressions were analyzed in 32 HSCR patients and 4 normal colons.
As revealed in Fig Fig2,2, the FHL1 mRNA relative expression in aganglionic colons was 1.06��0.49 (ganglionic colon relative expression level was 1) (P=0.44). FHL1 protein gray level relative to GAPDH in normal colons was 0.83��0.09. FHL1 expression level in ganglionic colon (1.66��0.30) or aganglionic colon (1.81��0.35) was significantly higher than that in normal colons Cilengitide (P=0.045 and P=0.041, respectively).