4.3. Western Blot Analysis

The perirenal fat pad (1 g) was homogenized with 3 mL of RIPA buffer solution (ddw 8.7 mL/10× RIPA buffer 1 mL/100 mM PMSF 100 µL/Phosphatase Inhibitor Cocktail2 100 µL/Phosphatase Inhibitor Cocktail3 100 µL) at 4 °C for 10 min. The homogenate was centrifuged (3000 rpm, 5 min, 4 °C) to obtain the liquid at the bottom, which was centrifuged (15,000× g, 20 min, 4 °C) to acquire the cell protein for western blot analysis.

Aliquots of the extract, each containing 40 μg of protein, were evaluated for the expression of IR, GLUT4, PKC-ζ, PFK, PK, and ATGL. The samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were electrotransferred to a polyvinylidene difluoride membrane that was incubated with blocking buffer (phosphate-buffered saline containing 0.05% Tween-20 (PBST) and 5% w/v nonfat dry milk) for 1 h, washed three times with PBST, and then probed with 1:1000–1:2000 diluted solutions of anti-IR, anti-GLUT4, anti-PKC-ζ, anti-PFK, anti-PK, and anti-ATGL at 4 °C overnight. The intensity of the blots probed with 1:2000 diluted solutions of anti-α-tublin was used as a control to ensure that a constant amount of protein was loaded into each lane of the gel. The membrane was washed three times for 5 min each in PBST, and then incubated in a solution of horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibody, washed three times again for 5 min each in PBST, and exposed to the enhanced chemiluminescence reagent (Millipore, Darmstadt, Germany). Autoradiography was scanned and analyzed using a UVP Biospectrum image system (Level, Cambridge, UK).