FIGURE 4.

NASP expression was negatively correlated with miR-29a levels in vivo. A, Northern blotting detection of miR-29a levels in postnatal epididymis of days 1, 7, 15, 30, 45, 60, and 90. U6 snRNA was used as a loading control. B, Western blotting detection of Nasp expression in postnatal epididymis of days 1, 7, 15, 30, 45, 60, and 90. β-Actin was used as a loading control. C, detection of miR-29a and Nasp expression in postnatal rat epididymis by in situ hybridization and immunohistochemistry, respectively. miR-29a and Nasp expression were measured in the rat epididymis of days 1, 7, 28, and 49 by staining the paraffin-embedded tissue sections. Probes specific for miR-29a were labeled with digoxigenin (DIG) and expression was visualized using alkaline phosphatase substrate. Positive-expressing miR-29a cells stain blue (arrow). The rabbit polyclonal antibody toward Nasp was used for immunohistochemistry and the secondary antibody was labeled with biotin, and the expression was visualized using horseradish peroxidase (HRP). The brown staining represents the positive immunoreactivity for the Nasp as indicated (arrowhead). All pictures were taken at an original magnification of ×400. D, Northern blotting detection of miR-29a (left), and Western blotting detection of NASP (right) in NIH3T3, PC-1, and HEK 293T cells. E, Northern blotting detection of miR-29a (left), and Western blotting detection of Nasp (right) in 60-day-old rat epididymis and testis. 5 S rRNA was used as a loading control for Northern blotting and β-actin was used as a loading control for Western blotting.