*4.8. Western Blotting*

Exponentially growing cells were lysed in lysis bu ffer (50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing 1 mM 4-(2-aminoethyl)-benzensulfonyl fluoride hydrochloride. The cell lysate was cleared by centrifugation at 15,000 rpm for 10 min at 4 ◦C, and then supernatant was used as the total cellular protein. Total protein concentration was determined by the BCA protein assay (Pierce, Rockford, IL). Protein samples (8 or 16 μg) were electrophoresed on SDS-polyacrylamide gel and were electrophoretically transferred to a polyvinyl difluoride membrane in a transfer bu ffer (100 mM Tris, 192 mM glycine). After overnight incubation with blocking solution (10% skim milk), the membrane was incubated with the primary antibodies, a biotinylated anti-mouse or anti-rabbit IgG antibodies, and streptavidine-alkaline phosphatase. The bands were visualized after addition of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as a substrate. The primary antibodies used in this study are anti-adiponectin (clone 19F1, Abcam Co. Ltd., Tokyo, Japan), anti-FABP4 (Abcam Co. Ltd., Tokyo, Japan), anti-PPARγ (clone 81B8, Cell Signaling technology Japan, Tokyo, Japan), and antiα/β-tubulin (Cell Signaling technology Japan).
