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. 2002 Aug 5;158(3):487–496. doi: 10.1083/jcb.200204048

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Copyright © 2002, The Rockefeller University Press

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Figure 1. — BubR1 is a phosphoprotein associated with Bub3. (A) Specificity of the anti-BubR1 antibody. Interphase (lanes 1 and 4), metaphase (lanes 2 and 5), or spindle checkpoint–active (lanes 3 and 6) extracts were immunoblotted with anti-BubR1 antibody (lanes 4–6) or the same antibody preincubated with recombinant BubR1 protein (lanes 1–3). The migration of molecular standards is indicated on the left. (B) Anti-BubR1 antibody recognizes BubR1, but not Bub1. Immunoprecipitation was performed from metaphase extracts with a control IgG (lanes 1 and 4), anti-Bub1 (lanes 2 and 5), or anti-BubR1 (lanes 3 and 6) antibody. The supernatants left after immunoprecipitation (lanes 1–3) or the immunoprecipitates (lanes 4–6) were probed with anti-BubR1 (top) or anti-Bub1 (bottom) antibody. The migration of molecular standards is indicated on the left. (C) BubR1 is a phosphoprotein. Interphase (I; lanes 1, 4, and 7), metaphase (M; lanes 2, 5, and 8), and spindle checkpoint–active (N; lanes 3, 6, and 9) extracts were treated with phosphatase buffer (lanes 1–3), LPP (lanes 4–6), or LPP and phosphatase inhibitors (lanes 7–9). The extracts were then immunoblotted with anti-BubR1 antibody. The migration of molecular standards is indicated on the left. (D) BubR1 associates with Bub3. Anti-BubR1 immunoprecipitates were prepared from interphase (lanes 1 and 5), metaphase (lanes 2 and 6), or spindle checkpoint–active (lanes 3 and 7) extracts, or from Bub1-depleted extract with the spindle checkpoint provoked (lanes 4 and 8). The extracts (lanes 1–4) or the immunoprecipitates (lanes 5–8) were immunoblotted with anti-BubR1 (top) or anti-Bub3 (bottom) antibody.