Journal: The Journal of Biological Chemistry
Article Title: The F-box Protein FBXO25 Promotes the Proteasome-dependent Degradation of ELK-1 Protein *
Figure Lengend Snippet: The degradation of ELK-1 is proteasome-dependent and mediated by FBXO25. The methionine chase labeling experiments were performed using HEK293T cells transfected with the indicated plasmids. After the indicated times, the supernatants were obtained and submitted to immunoprecipitation ( IP ) with anti-ELK-1 antibody. The immunoprecipitated proteins were visualized by Coomassie Blue, and dried gels were film-exposed to protein visualization. A , FBXO25 and not pcDNA3 mediated ELK-1 degradation. B , 35 S-labeled ELK-1-FLAG-His 6 is degraded in the presence of FBXO25 after 12 h of transfection but not by pcDNA3 as indicated in densitometry graphics. C , 35 S-labeled ELK-1 degradation mediated by FBXO25 is proteasome-dependent. D , the proteasome inhibitor epoxomicin protected 35 S-labeled ELK-1-FLAG-His 6 of degradation by FBXO25 as indicated in densitometry graphics. For all experiments n = 2. H.C ., heavy chain. *, the apparent molecular mass of the radiolabeled band ELK-1-FLAG-His 6 on the gels was ascertained by comparison with the mobility of FLAG affinity-purified ELK-1-FLAG-His 6 (data not shown).
Article Snippet: After 24 h, the cells were washed with PBS and DMEM without methionine (Sigma-Aldrich) and kept in this medium for 2 h. Then the cells were pulsed for 1 h with 150 μCi/well 35 S-radiolabeled methionine (Express labeling mix 35 S; PerkinElmer Life Sciences).
Techniques: Labeling, Transfection, Immunoprecipitation, Affinity Purification