Figure 4.

Effect of rosiglitazone on c-Cbl tyrosine phosphorylation in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes were treated with or without 5 μM rosiglitazone for 12 h before serum starvation for an additional 12 h in the presence of rosiglitazone. After insulin stimulation for 1 min, equal amounts of protein were immunoprecipitated with anti-c-Cbl antibodies. The resulting immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine antibodies (Anti-pY Blot). The blot then was stripped and reprobed with anti-c-Cbl antibodies (Anti-c-Cbl Blot). (B) The Western blots from A were quantitated by densitometric scanning. The results represent the intensities of the signal corresponding to tyrosine-phosphorylated c-Cbl normalized against that of c-Cbl protein in the same sample. (C) Equal protein amounts (30 μg per lane) of the cell lysates used in A were analyzed directly by antiphosphotyrosine antibodies immunoblotting (anti-pY Blot). The positions of the insulin receptor (IR) and the insulin receptor substrate 1 (IRS-1) are indicated by arrows. Results are representative of experiments performed at least three times.