FIGURE 4.

A β-agonist stimulates Fsp27 accumulation and increases Fsp27 stability. A, oleic acids (OA) promote Fsp27 accumulation. Differentiated 3T3-L1 adipocytes (8 days after induction) were treated with OA (400 μm) for 0, 2, 6, 12, or 24 h, and the Fsp27 protein level was analyzed. β-Actin was used as a loading control. B, Iso stimulates Fsp27 accumulation. Differentiated 3T3-L1 adipocytes were treated with Iso (10 μm) for the indicated amount of time (0, 0.5, 1.5, or 3 h), and the Fsp27 level was evaluated. Perilipin A was used as a control for the LD-associated protein. Tubulin was used as a loading control. C, Iso enhances Fsp27 stability. Differentiated 3T3-L1 adipocytes were treated with CHX in the presence or absence of Iso. Cells were harvested 0, 30, 60, or 90 min after the addition of Iso and CHX. D, the quantitative analysis of the Western blot in C demonstrated that Fsp27 had an increased half-life in the presence of Iso. Experiments were repeated three times. ***, significant difference from control curve using a two-way ANOVA, p < 0.001. E, reduced Fsp27 ubiquitination in 3T3-L1 adipocytes treated with Iso. Differentiated 3T3-L1 adipocytes were treated with MG132 alone or together with Iso for 2 h. Fsp27 was immunoprecipitated, and the ubiquitination level was determined using a ubiquitin-specific antibody. F, kinetic analysis of the Fsp27 level after Iso withdrawal in 3T3-L1 adipocytes. Cells were harvested at various time points after Iso treatment and withdrawal. Iso was washed away from the culture medium 2 h after its addition. G, OA enhanced Iso-induced Fsp27 accumulation. Differentiated 3T3-L1 adipocytes were treated with Iso alone or together with OA for 1 h, and the Fsp27 protein level was analyzed using Western blot. H, quantitative analysis of the Fsp27 level in G. A Student's two-tailed t test (unpaired) was used for statistical analysis (***, p < 0.001; **, p < 0.01, n = 3).