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The subcellular localization of SF-1

The subcellular localization of SF-1. Fu Huang. Lab Introduction. Lab 429, Institute of Molecular Biology, Academia Sinica. Research interests I. Steroid Function and Regulation II. Zebrafish Development. PI: Bon-chu Chung, Ph.D. Sumoylation regulates nuclear distribution of SF-1

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The subcellular localization of SF-1

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  1. The subcellular localization of SF-1 Fu Huang

  2. Lab Introduction Lab 429, Institute of Molecular Biology, Academia Sinica Research interests I. Steroid Function and Regulation II. Zebrafish Development PI: Bon-chu Chung, Ph.D.

  3. Sumoylation regulates nuclear distribution of SF-1 • Cytoplasmic distribution of SF-1.

  4. Schematic overview of the biosynthetic pathways for steroid hormones SF1 regulates the expression of P450 steroid hydroxylases.

  5. Steroidogenic factor 1 (SF-1) SF-1 • The Ftz-F1 box of SF-1 contains NLS sequence. (Li et al., 1999) • SF-1 is an orphan nuclear receptor. • SF-1 was always found in nucleus in previous studies.

  6. SUMO-1 SF-1 • SUMO-1 is a 101 amino acid polypeptide and only 1-97 a.a. can be attached to target proteins. • SF-1 has two sumoylation sites, K-119 and K-194. • SUMO-1 can trigger the formation of PML nuclear bodies.

  7. Cloning of FLAG-RFP-SUMO-1 fusion protein SUMO-1 WT: 94 95 96 97 CAA ACG GGGGGT Gly Gly SUMO-1 AA (mutant): 94 95 96 97 CAA ACG GCGGCT Ala Ala

  8. Western blot of FLAG-RFP-SUMO-1 WT AA Sumoylated proteins FLAG-RFP-SUMO-1

  9. Sumoylation regulates nuclear distribution of SF-1 RFP-SUMO-1 WT SF-1-GFP Merge WT RFP-SUMO-1 AA SF-1-GFP Merge AA

  10. Sumoylation regulates nuclear distribution of SF-1 • Cytoplasmic distribution of SF-1.

  11. Cytoplasmic distribution of SF-1 SF-1 Ab SF-1-GFP Phase Merge

  12. Cytoplasmic distribution of SF-1 RFP SF-1-GFP 3Dview Merge

  13. Cytoplasmic localization of SF-1 Actin Ab SF-1-GFP Merge 3D view

  14. Cytoplasmic localization of SF-1 Tublin Ab SF-1-GFP Merge 3D view

  15. Cytoplasmic localization of SF-1 SCC Ab SF-1-GFP Merge

  16. LMB does not affect cytoplasmic distribution of SF-1 15min 45min 1h15min 1h45min 2h15min 2h45min 3h15min 3h45min 4h15min 4h45min 5h15min 5h45min 6h15min 6h45min (After LMB treatment)

  17. SF-1 contains LMB- insensitive NES homologue (306-381) SF-1 (359-436) FF1b (743-817) AR (306-381) SF-1 (359-436) FF1b (743-817) AR (743-817) AR (809-883) MR (386-455) ER (743-817) AR (809-883) MR (Saporita et al., 2003) (386-455) ER

  18. The LBD domain of SF-1 contains LMB-independent NES homologue SF-1

  19. Future work • Examine the interaction of SF-1 and SUMO-1 in living cell under different stimulus. • Examine the candidate NES fragment of SF-1. • Use electro-microscopy to find out the exact location of cytoplasmic SF-1.

  20. Acknowledgments • Dr. Bon-chu Chung • Mr. Wei-yi Chen • Mr. Wen-chih Lee • Ms. Lee • All the lab members

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