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Fig. 3. SKP1-CUL1-βTrCP is the E3 ubiquitin ligase that mediates the UV-induced degradation of securin. (A) COS-7 cells transiently transfected with pCDNA3 or pCDNA3-Flag-CUL1_1-452 were irradiated (UV) or left untreated (C) 1 hour 30 minutes before harvesting. NP40 extracts were prepared and western blotted for CUL1, Flag-CUL1_1-452 and securin detection. (B) COS-7 cells were transiently transfected with the indicated plasmids and treated as above. Western blots were probed for the presence of CUL1, Flag-CUL1_1-452, securin-VSV and EGFP. The asterisk indicates an nonspecific band. (C) COS-7 cells were transiently transfected with pCDNA3 or pCDNA3-MT-Skp2 ${Delta}$ F, and irradiated (UV) or left untreated (C) 1 hour 30 minutes before harvesting. Extracts were western blotted for SKP2, MT-SKP2 ${Delta}$ F, and securin detection. (D) COS-7 cells were transfected with the indicated plasmids, treated as described, and blotted with anti-SKP2, anti-VSV, and anti-GFP. (E) COS-7 cells transiently transfected with pCS2-HA or pCS2-HA-βTrCP ${Delta}$ F were left untreated (C), irradiated (UV), or treated with okadaic acid (OA) 1 hour 30 minutes before harvesting. NP40 extracts were prepared and western blotted for the detection of the indicated proteins. Cells treated with OA were used as a negative control of the experiment because phosphorylated forms of securin after OA treatment are degraded via the SCF complex (Gil-Bernabe et al., 2006), but βTrCP ${Delta}$ F does not prevent this degradation. (F) Transfected COS-7 cells with the indicated plasmids were treated as described, and blotted for the presence of HA-βTrCP ${Delta}$ F, securin-VSV, and EGFP.

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