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. 2001 May 14;153(4):649–662. doi: 10.1083/jcb.153.4.649

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© 2001 The Rockefeller University Press

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Bul1p and Bul2p specify polyubiquitination of Gap1p. Wild-type (CKY703; lanes 1–3) and bul1Δ bul2Δ (CKY704; lanes 4 and 5) strains were transformed with combinations of empty vector (pRS316; lane 1) or vector-carrying HA-Gap1p, (pCK227; lanes 2–5), and either pP_CUP1-UBI-c-myc (pCK231; lanes 1, 3, and 5) or pP_CUP1-UBI (pCK232; lanes 2 and 4). Strains were cultured overnight in minimal medium (SD Urea) at 24°C, and CuSO₄ was added to 0.1 μM to induce the CUP1 promoter 3 h before harvesting in exponential phase. Anti-HA (3F10) immunoprecipitates were subject to SDS-PAGE and Western blotting with either (A) anti-HA (16B12) antibody or (B) anti–c-myc (9E10) antibody. m, monoubiquitinated HA-Gap1p; p, polyubiquitinated HA-Gap1p. (C) Lanes 1 and 2 correspond to lane 5 from A and B, respectively, except that sixfold more yeast extract was used for immunoprecipitation and Western blotting.