Activin-lnduced Inhibin: MATERIALS AND METHODS(1)

9 Dec
2012

MATERIALS AND METHODS(1)

Hormones and Reagents

Ovine FSH (NIH-FSH-S20, activity 175 X NIH-FSH-Sl) and a monoclonal antibody against IGF-I (anti-IGF-I antibody) were kindly provided by the National Hormone and Pituitary Program (NIDDK). A specific Inh-a antibody, kindly provided by Dr. Nicholas Ling (Neurocrine Biosciences, San Diego, CA), was generated in the rabbit against an amino terminal 30-amino acid chain of porcine Inh-a coupled to BSA. Recombinant human follistatin-315 (FS-315) was prepared as previously described. Recombinant human IGF-I was from GroPep (Adelaide, Australia). Tyrphostins A23 and A63, and horseradish peroxidase-conjugated goat anti-rabbit IgG were from Calbio-chem (San Diego, CA). Androstenedione, diethylstilbestrol (DES), control mouse ascites fluid, and chloramine T were from Sigma (St. Louis, MO). Fetal calf serum, McCoy’s 5a medium, L-glutamine, penicillin, and streptomycin sulfate were from Gibco (Santa Clara, CA). 125I-labeled sodium and [a-32P]uridine triphosphate (UTP) were from Amer-sham (Arlington Heights, IL). ventolin inhaler

Recombinant Protein Expression

Recombinant rat IGF binding protein (IGFBP)-5 and recombinant rat activin A were expressed in Chinese hamster ovary (CHO) cells under the control of the simian virus-40 promoter essentially as described using the full-length rat IGFBP-5 cDNA clone, pRBP5-506, and rat inhibin (3A-subunit cDNA, (5АЗО, respectively. CHO cells produce their own IGFBP-4 (Shimasaki, unpublished result), and thus, the secreted IGFBP-4 was purified from the conditioned medium of CHO cell cultures. IGFBPs and activin A were purified using IGF-I and follistatin affinity columns, respectively, and then two steps of reverse-phase HPLC. The purity and quantification of these proteins were analyzed by amino acid analysis and N-terminal amino acid sequence analysis.

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