Figure 1
Serum starvation induces ubiquitin-dependent degradation of SF-1.
(a) Decrease in SF-1 levels after 4 hours of serum starvation. HEK293 cells transfected with SF-1 were incubated in serum-free medium and analyzed by Western blot using an anti-SF-1 antibody (*P < 0.05). (b) Temporal regulation of SF-1 after starvation. SF-1-expressing cells were incubated in serum-free medium for the indicated times and the SF-1 levels were analyzed by Western blot using an anti-Flag antibody (*P < 0.05). (c) Changes in SF-1 levels after serum starvation in organotypic VMH slices. Hypothalamic organotypic slices were incubated in standard medium or serum-free medium for 6 hours and then analyzed using an anti-SF-1 antibody (**P < 0.01). (d) Protein turnover of SF-1 under different nutritional conditions. HEK293 cells were transfected with Flag-tagged SF-1 and treated with CHX (30 mg/ml) with or without serum (*P < 0.05). (e) Degradation of SF-1 by the ubiquitin-dependent proteasome system. Cells were treated with or without MG132 at 25 μM for 6 hours in normal or serum starvation conditions (*P < 0.05). (f) Increased SF-1 ubiquitination in the serum-starvation condition. SF-1 was immunoprecipitated using an anti-SF-1 antibody followed by Western blot using either the anti-HA or anti-SF-1 antibodies. The values are presented as the mean ± SEM from three independent experiments (*P < 0.05).
