**2.4 Protein extraction and western blot**

Frozen kidneys were mixed at 4°C with a Turrax homogenizer at 100 mg frozen tissue per 2 mL of lysing buffer containing protease inhibitor (Laemmli, 1970). After centrifugation, the concentration of soluble proteins was determined using the Bradford protein assay (Bradford, 1976). Fifty or 100 µg samples of soluble proteins were subjected to 10% PAGE containing 0.1% SDS using 6 Watts per gel. Protein transfer and equal loading of proteins were visualized on the membranes with Ponceau S solution. Blocked immunoblots were incubated with primary polyclonal rabbit antibodies raised against rat/mouse-OAT (dilution 1:1,000), rat-aldose reductase (AR; EC 1.1.1.21, dilution 1:3,000) (Lambert-Langlais et al, 2009), and monoclonal mouse β-actin (dilution 1:2,000) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH; EC 1.2.1.12, dilution 1:170) antibodies, and then with a peroxidase-conjugated anti-rabbit or antimouse IgG secondary antibodies (dilution 1:10,000) as previously described (Levillain et al., 2004, 2005). Antibody binding was revealed using chemiluminescence (ECL) Western Blotting Kit. Details for ECL detection and quantitation of the bands have been mentioned earlier (Levillain et al., 2004, 2005).

Orchidectomy Upregulates While Testosterone Treatment Downregulates

significantly in our experimental conditions (data not shown).

agreement with published data (Levillain et al., 2007; Ventura et al. 2009).

Fig. 2. Technical verifications to determine the conditions for OAT assay.

the coefficient of correlation was statistically significant (*P* < 0.01 or less).

**3.2 Influence of orchidectomy on the expression of OAT gene** 

Closed circles: female mouse kidney and closed triangles: male mouse kidney. In all cases,

A complete analysis of the expression of OAT gene in the mouse kidney was undertaken by analyzing the levels of mRNA and protein, and measuring OAT activity. The level of OAT

**3. Results** 

**3.1 Technical verifications 3.1.1 Semiquantitative RT-PCR** 

**3.1.2 OAT activity assay** 

the Expression of Ornithine Aminotransferase Gene in the Mouse Kidney 121

To quantify the levels of OAT and cyclophilin A mRNA by RT-PCR, we determined the appropriate number of amplification cycles to remain in the exponential phase of the amplification process and avoid saturation (Table 1). For each mRNA studied a single RT-PCR product was obtained. The amplified RT-PCR products were of the expected size (Table 1). The level of the mRNA of a housekeeping gene (cyclophilin A) did not vary

The renal cortex of male and female mice was dissected and prepared to quantify OAT activity. To determine the conditions for assay of OAT activity, the incubation time was tested every 5 min up to 30 min and the protein concentration varied from 0 to 400 µg per sample. OAT activity increased linearly with the concentration of soluble proteins up to 400 µg in male mouse cortex and 300 µg in that of the female when the incubation time was fixed at 30 min (Fig.2 left, male: r2=0.99809 and female: r2=0.99583, *P* < 0.01). OAT activity increased linearly in proportion with the incubation time up to 30 min when the concentration of soluble proteins was fixed at 140 µg in both male and female mice (Fig. 2 right, male: r2=0.99352 and female: r2=0.9972, *P* < 0.01). Constantly, OAT activity was more than three-fold higher in female than in male mouse kidney. The sex-differential OAT activity found in the present results is in good
