FIG. 3.

Bop1 is the core factor of the PeBoW complex. (A) U2OS cells were transfected twice with the indicated siRNAs. Endogenous protein levels were analyzed by Western blotting 2 days after the last transfection. α-Tubulin is shown as a loading control. (B) Cells were treated as described for panel A and metabolically labeled with [³²P]orthophosphate for 60 min 1 day after the last transfection. Subsequently, cells were incubated for 2 h in regular culture medium. Labeled rRNAs are indicated. Ethidium bromide staining is shown as a loading control. (C) Total cell lysates of U2OS cells transfected twice with the indicated siRNAs were separated by native gel electrophoresis 1 day after the last transfection. Endogenous Pes1, Bop1, and WDR12 were visualized by immunoblotting. Nonincorporated proteins or complexes containing the respective factor are indicated. (D) U2OS cells were transfected at days 0 and 1 with either control or Bop1-specific siRNA. Total cell lysates were subjected to immunoprecipitation with antibodies either against Pes1 (8E9), Bop1 (6H11), WDR12 (1B8), or the isotype control coupled to protein G-Sepharose beads 2 days after the last transfection. Equivalent amounts of total lysates used for immunoprecipitation (IP) were loaded in each lane or 20% thereof for the input. Immunodetection was performed with the anti-Pes1 (8E9) or anti-WDR12 (1B8) antibody, respectively. An asterisk indicates cross-reactivity to immunoglobulin molecules.