A Possible Role for the Pentose Phosphate Pathway: MATERIALS AND METHODS(6)

Immunoblotting

Western analysis was performed using procedures similar to those described for the detection of GLUT in preimplantation embryos by Aghayan et al.. Briefly, 0.25 X 106 spermatozoa or 30 mouse blastocysts were lysed in 10 ^l of lysis buffer and stored at-20°C. Before electrophoresis, the samples were diluted in a double-strength solution of Laemmli’s sample buffer. Electrophoresis was performed under reducing conditions using a 10% polyacrylamide SDS minigel system (Bio-Rad, Zurich, Switzerland), and the proteins were transferred to the nitrocellulose membrane with the Bio-Rad mini-transfer system. After transfer, the membrane was washed and prepared, using a slight modification of the procedure described by Aghayan et al.. To detect GLUT1 and GLUT3, respectively, the membrane was incubated in anti-GLUT1 (1: 1500 dilution) or anti-GLUT3 (1:1500 dilution) for 1 h at room temperature. Proteins were visualized by using the enhanced chemiluminescence system (ECL; Amersham). buy cheap antibiotics

Statistics

ANOVA followed by Scheffe’s test for multiple comparisons was used to compare the numbers of bound sperm, the numbers of decondensed sperm, and glucose metabolism in the different media. The percentages were compared using the same method but after arc sin transformation.

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