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. 2007 Mar 12;27(10):3682–3694. doi: 10.1128/MCB.00172-07

FIG. 2.

FIG. 2.

(A) Overexpression of Bop1 reduces the endogenous Bop1 protein level. H1299 cells were stably transfected with the indicated constructs. After 6 days, endogenous Pes1, Bop1, and WDR12 protein levels were analyzed by Western blotting. Human-specific anti-Bop1 MAb 6H11 does not recognize recombinant mouse Bop1. Expression levels of HA-tagged proteins were determined with anti-HA antibody 3F10. Equal loading was verified by immunodetection of α-tubulin. (B) Overexpression of Bop1 titrates endogenous WDR12 into a Bop1/WDR12 subcomplex. Total cell lysates of U2OS cells expressing the indicated proteins were separated by native gel electrophoresis after 1 day of expression. Pes1, Bop1, and WDR12 were visualized by immunoblotting. Nonincorporated proteins or complexes containing the respective factor are indicated. (C) Overexpression of Bop1-HA replaces endogenous Bop1 in PeBoW complex formation. Total lysates of U2OS cells expressing luciferase or Bop1-HA were separated by native gel electrophoresis after 6 days of expression. Pes1, Bop1, WDR12, and Bop1-HA were visualized by immunoblotting. An asterisk indicates monomeric Bop1-HA. Double asterisks indicate the Bop1/WDR12 subcomplex. (D) Total lysates of U2OS cells expressing the indicated proteins were separated by native gel electrophoresis. WDR12 were visualized by immunoblotting. Nonincorporated proteins or complexes containing the respective factor are indicated. Expression levels of HA-tagged proteins were determined by Western blot analysis with anti-HA antibody 3F10. Equal loading was verified by immunodetection of α-tubulin. (E) Cytoplasmic and nuclear fractions of H1299 cells expressing the indicated genes were analyzed by native gel electrophoresis as described for panel B. Complexes were visualized by immunostaining with HA-specific antibodies.