FIG. 6.

Adiponectin reduces PKA RIIα protein stability. 3T3-L1 cells were differentiated into adipocytes. A: Adipocytes were co-cultured overnight with Ad-Acrp30–transduced FAO cells. Cycloheximide (100 μg/mL) was added to the medium, and the insert wells with FAO cells were removed. Protein samples of 3T3-L1 adipocytes were prepared at the indicated time points. B: 3T3-L1 adipocytes were treated with adiponectin-enriched conditioned medium from Ad-Acrp30–transduced FAO cells. C: Proteasome inhibitor MG132 (25 μmol/L) was added to the medium 30 min before adiponectin treatment. Protein samples were prepared 2 h later. D: Mature 3T3-L1 adipocytes were co-cultured with adiponectin-expressing FAO cells for 4 h. The Pierce Crosslink IP kit (Thermo Scientific Pierce Protein Research Products, Rockford, IL) was used for immunoprecipitation to eliminate nonspecific bands. All studies were repeated 4–6 times. The quantified data are expressed as means ± SEM. *P < 0.05 vs. control cells at the same time point (B) or without MG132 treatment (C).