Gonadotropin-Independent Regulation of Steroidogenesis: MATERIALS AND METHODS(7)

8 Nov

Southern Hybridization Analysis

The cDNA fragments generated by RT-PCR were resolved on 1.2% agarose gel and transferred onto nylon membranes (Hybond-N; Amersham). The membranes were prehybridized for at least 1 h at 65°C in a total volume of 25 ml containing 5-strength SSPE (single-strength SSPE = 180 mM NaCl, 10 mM sodium phosphate, and 1 mM EDTA, pH 7.7), 5-strength Denhardt’s solution, 0.5% (w: v) SDS, and calf thymus DNA (20 |xg/L). Hybridization was performed at 65°C overnight in the prehybridization solution to which the 32P-labeled cDNA (nucleotides 186— 364 of rat LH|3 cDNA) probe was added. The cDNA probe was labeled by using a Multiprime DNA labeling system (Amersham) with [a-32P]dCTP (3000 Ci/mmol; Amersham). The blots were washed twice for 10 min in doublestrength SSPE-0.1% SDS at room temperature and once in single-strength SSPE-0.1% SDS at 65°C for 30 min. The x-ray films were exposed for 4 days. ventolin inhalers

Statistical Analysis

All values are means ± SEM. A Macintosh version of the superANOVA program (Abacus Concepts, Inc., Berkeley, CA) was used for 1-factor ANOVA, followed by Duncan’s New Multiple Range and Fisher’s protected LSD post-hoc tests. A p value less than 0.05 was chosen as the limit of statistical significance.