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. 2007 Jul 13;405(Pt 3):495–501. doi: 10.1042/BJ20061886

Figure 4. XEF1AO is a Scythe-associated protein that is polyubiquitinated in a Scythe-dependent manner in cultured cells.

Figure 4

(A) 2S-tagged full length Scythe and 3T7-tagged XEF1AO were co-expressed in HeLa cells. Cells were incubated with or without 20 μM MG132 for 6 h before being harvested as indicated. Tagged proteins were affinity-purified with S-protein–agarose beads, and bound proteins were immunoblotted with either anti-S-peptide or anti-T7-tag antibodies. (B) XEF1AO, which was associated with Scythe, is covalently linked with ubiquitin. FLAG-tagged Scythe and 2S-tagged XEF1AO were expressed as in (A). Tagged proteins were immunoprecipitated with anti-FLAG M2– agarose beads, and the precipitates were eluted by SDS denaturation buffer. The eluted samples were diluted with RIPA buffer that did not contain SDS, and then the samples were affinity-purified with S-protein–agarose beads (second precipitation). Bound proteins were subjected to Western blot analysis with anti-ubiquitin, anti-S-peptide and anti-FLAG antibodies. ppt, precipitation.