X omat ar

A protocol of a pre�ious study was used for the preparation of cell lysates (14) In brief, the proteins (30 µg) were resol�ed using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Protogel, Atlanta, GA, USA) and transferred onto poly�inylidene difluoride nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The filters were incubated at room temperature for 2 h in 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.1% (�/�) Tween 20, 0.5% (w/�) bo�ine serum albumin (TBST/BSA) and then overnight at 4˚C on a shaker with the rabbit monoclonal anti-GAPDH (ab181602) and anti-CTGF (ab6992) antibodies (1:1,000 in TBST/BSA; Abcam, Cambridge, UK). The membranes were washed twice with TBST and incubated for 2 h with the horseradish pero�idase-conjugated secondary antibody (1:20,000 in TBST/BSA). Following the final washes, the signals were visualized with Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Healthcare Life Sciences) and autoradiographic film (X-Omat AR; Kodak, Rochester, NY, USA). Densitometric analysis using ImageQuant software was performed following digital scanning (Odyssey ® Fc; Agfa-Ge�aert, Mortsel, Belgium).

For Southern blot analysis, approximately 5 to 15 µg of genomic DNA, extracted as previously reported, were digested with specific restriction enzymes, were separated on a 1% (wt/vol) agarose and Tris-acetate-EDTA gel and were blotted onto a Hybond NX membrane (Amersham Biosciences, Milano, Italy). Digoxygenin (DIG)-labeled specific probes were generated by PCR with specific primers, using genomic or plasmid DNA as template, and were used for overnight hybridization at 65°C. The PCR reactions were performed in a volume of 25 µl, using DIG-11-dUTP (Roche, Mannheim, Germany), and consisted of a denaturation step at 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min. Southern hybridization and detection of the DIGlabeled probes were performed according to manufacturer's instruction. Membranes were exposed to X-ray film (X-Omat AR, Kodak, Rochester, NY, U.S.A.) for approximately 1 to 3 h.

After each treatment, CHO-K1/A5 cells were washed with M1 and lysed at 4 • C during 1 h with cell lysis buffer (20 mM HEPES; 10 mM EGTA; 5 mM ␤-glycerol phosphate, 1% Nonidet P-40; 2.5 mM MgCl 2 ) containing protease inhibitors (2 g/ml leupeptin; 1 g/ml aprotinin; 1 g/ml pepstatin; 0.1 mM PMSF). Proteins (40 g) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Membranes were blocked and probed with primary antibodies overnight at 4 • C followed by incubation with the corresponding secondary antibodies at room temperature for 2 h. Immunoreactive bands were detected by enhanced chemiluminescence reagent (ECL, Amersham Biosciences) using standard X-ray film (Kodak X-Omat AR).

Adipocytes were homogenized by using a Dounce homogenizer in a buffer containing 10 μg/ ml aprotinin, 1 mM EDTA, 20 mM HEPES (pH 7.4), 5 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 0.25 mM sucrose. PM fractions were prepared as described previously (27) .
Western blot analysis PM fractions prepared from aliquots of adipocyte homogenates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a Hybond-enhanced chemiluminescence (ECL) filter (GE Healthcare Life Sciences). The separated proteins transferred onto the filter were incubated for 1 h with anti-GLUT4 rabbit antibody (1:1000, carboxy-terminal polypeptide [27] ). After incubating with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin-G antibody (1:2500; GE Healthcare Life Sciences), the immune complexes were detected on X-ray film (Kodak X-Omat AR; Eastman-Kodak Co., Rochester, NY) by using an ECL detection system (GE Healthcare Life Sciences). The blots on the film were quantified using the National Institutes of Health Image program (http://rsb.info.nih.gov/nih-image/).