FIGURE 3.

Ubiquitination and proteasome-mediated degradation. A, ubiquitination is shown. HA-SIRT1 was expressed in 293 cells and collected after JNK activation using the anti-HA antibody through IP. Ubiquitination of the SIRT1 protein was determined using the ubiquitin antibody. IB, immunoblot. B, proteasome effect is shown. The proteasome inhibitor MG132 (50 μm) was used to pretreat 3T3-L1 adipocytes for 30 min. SIRT1 degradation was induced by insulin (200 nm, 2 h). Phosphorylation of Akt Ser-473 was used as a control of insulin signaling. C, SIRT1 protein stability is shown. WT and JNK1-KO MEF cells were compared for SIRT1 protein half-life. Protein synthesis was inhibited with cycloheximide. SIRT1 protein abundance was measured in whole cell lysates at multiple time points (h).