MATERIALS AND METHODS(3)

RIA

The concentrations of testosterone in oRTF preparations were determined using an ImmuChem testosterone 125I RIA kit (ICN Biomedicals, Costa Mesa, CA) according to the manufacturer’s protocol. asthma inhalers

GGT Assay

GGT catalytic activity assay was performed as described previously . Protein concentrations of samples were determined by the Bio-Rad (Richmond, CA) protein assay. For the comparative study of GGT catalytic activity among various tissues and of the effects of EDL on the GGT catalytic activity in these tissues, GGT catalytic activity was expressed as nmol/min per milligram protein. For the observation of whether RTF preparations and bFGF could recover GGT catalytic activity, the GGT catalytic activity was expressed as percentage of GGT catalytic activity in relation to that of each sham control.

Western Blot Analysis

Equal amounts of proteins were fractionated by 12% SDS-PAGE and transferred to nitrocellulose membranes (MSI). As a protein loading control, the membranes were stained with Ponceau S to determine the efficiency of the protein loading and transfer . Immunoblotting was not performed on membranes if different protein levels were seen in the different lanes as indicated by the stain. For immunoblotting with GGT antibody, the filters were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween (TBST: 150 mM sodium chloride, 10 mM Tris-hydrochloride, pH 8.0, 0.05% [v:v] Tween 20) and then incubated with a GGT polyclonal antibody (pAb; generously provided by Dr. Yannick Laperche [INSERM 99, Creteil, France]). After being washed twice with TBST, the filters were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz Biotech, Santa Cruz, CA).