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. 2007 Jul;18(7):2429–2440. doi: 10.1091/mbc.E07-01-0011

Figure 6.

Figure 6.

Requirements for ubiquitination of Tre1. (A) Immunoprecipitation of GFP-tagged Tre1Δlumenal or Tre1 Δlumenal PPAG with protein A-tagged Rsp5 from Δpep4 cells. Eluates from IgG-Sepharose beads enriched by a factor of 20. (B) Western blot, probed with anti-S-tag, of in vitro ubiquitination of recombinant Tre1Δlumenal (and Tre1 Δlumenal PPAG) by wild-type recombinant Rsp5 (WT) or Rsp5 carrying ΔWW1, ΔWW2, ΔWW3, ΔWW1,2, ΔWW1,3, or ΔWW2,3 mutations described above. (C) Western blot, probed with anti-S-tag, of in vitro ubiquitination of recombinant S-tagged TreΔlumenal PPAG by Rsp5 and Tap-purified wild-type Bsd2 or Tap-purified Bsd2 carrying ΔPY1, ΔPY2, 113-8, or GVG mutations described above. Arrows indicate mono-ubiquitinated forms of TreΔlumenal PPAG. (D) Western blot, probed with anti-S-tag, of in vitro ubiquitination of recombinant S-tagged TreΔlumenal PPAG mediated by Tap-purified Bsd2 and either wild-type Rsp5 or Rsp5 carrying ΔWW1, ΔWW2, ΔWW3, ΔWW1,2, ΔWW1,3, ΔWW2,3, ΔC2 mutations described above. Arrows indicate monoubiquitinated forms of TreΔlumenal PPAG. Irrelevant lanes in C and D have been excised, as indicated by the gaps, but exposures are identical for all lanes.