ZFIN Image: Ju Shin et al., 2015, Fig. 1

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Fig. 1

FACS and microscopic analyses of H2B-GFP expression after transient transfection of synthetic E3 ligase candidates.

(a) FACS analysis of H2B-GFP/293TetOn stable cell line transiently transfected with various synthetic E3 ligase candidates. After transfection, Myc₁₀-TM (transmembrane form of Myc₁₀ epitope) and each candidate ligase were simultaneously expressed from a bi-directional tetracycline response element (TRE) promoter following doxycycline treatment (Supplementary Fig. 2). vhhGFP4 is a nanobody against GFP. NSnoFbox was derived from NSlimb by deleting its F-box domain necessary for binding to the adaptor protein in the Skp1-Cullin1-F-box E3 ubiquitin ligase complex⁹. ‘–’ represents expression of Myc₁₀-TM without any candidate E3 ligase. Only vhhGFP4-SPOP E3 ligase greatly depleted H2B-GFP in the cells expressing Myc₁₀-TM. (b) Microscopic analysis of H2B-GFP/U2OS stable cell line transiently transfected with the ligase candidates. Doxycycline (1 µg/ml) was administered for 24 hours to promote expression of TagRFP and each ligase candidate. Each panel shows bright field, TagRFP (red), H2B-GFP (green), and merged images of both TagRFP and H2B-GFP. Only vhhGFP4-SPOP or vhhGFP4-SPOPΔNLS has no yellow nuclear signal in merged images because of H2B-GFP depletion in cells expressing TagRFP.

Acknowledgments

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