Molecular analyst

The respective RNA bands were analysed using a model GS-700 Imaging Densitometer and Molecular Analyst software (version 1.5; Bio-Rad Laboratories, Hercules, CA, USA).

Approximately 20 mg of frozen rat kidney tissues was homogenised in 200 µl of RIPA buffer and then centrifuged at 12,000 rpm for 10 min. The protein concentrations of the supernatants were measured according to the BCA method. Total proteins were denaturalized in boiling water for 5 min. Equal amounts of total protein were separated onto 10% SDS-PAGE and electrophoretically transferred to a polyvinylidenedifluoride (PVDF) membrane (Millipore, Shanghai, China) which preactivated with methanol in the transferring buffer. Membranes were blocked with 5% skim milk for 2 h and incubated overnight with specific primary antibodies at 4°C. Immunoreactive bands were detected using HRP-conjugated goat anti-rabbit IgG as the secondary antibody (1:5000) (Jackson, Shanghai, China). Immunoreactive bands were visualised using a phototope-horseradish peroxidase Western blotting detection system (Cell Signaling Technologies, Beverly, MA) and quantified through densitometry with Molecular Analyst (Bio-Rad Laboratories, Hercules, CA). Primary antibodies included rabbit polyclonal antibodies against rOAT1 (dilution 1:1000) and rOAT3 (dilution 1:500).

COX-2 and perilipin-2 protein levels in cultured macrophages were detected by Western blotting. Aliquots of MT-III-stimulated and non-stimulated cells (1.5×10⁶ cells) were lysed with 100 μL sample buffer (0.5 M Tris-HCl, pH 6.8, 20% SDS, 1% glycerol, 1 M β-mercapto ethanol, 0.1% bromophenol blue) and boiled for 10 min. Samples were resolved by SDS-PAGE on 10% bis-acrylamide gels overlaid with a 5% stacking gel. Proteins were then transferred to nitrocellulose membranes using a Mini Trans-Blot (Bio-Rad Laboratories, Richmond, CA, USA). The membranes were blocked for 1 h with 5% nonfat dry milk in Tris-buffered saline (TBS) (20 mM Tris, 100 mM NaCl, and 0.5% Tween 20, pH 7.2), and incubated with primary antibodies against COX-2 (1∶1000 dilution), perilipin-2 (1∶2000) and β-actin (1∶3000) for 1 h. They were then washed and incubated with the appropriate secondary antibody conjugated to horseradish peroxidase. The immunonereactive bands were detected by the entry-level peroxidase substrate for enhanced chemiluminescence (ECL), according to the instructions of the manufacturer (GE Healthcare). Band densities were quantified with a GS 700 densitometer (Bio- Rad Laboratories) using the image analysis software Molecular Analyst (Bio-Rad Laboratories).