FIGURE 2.

Identification of p160 as MYBBP1A. A, Aurora A phosphorylates p160 in nuclear extracts. Asynchronous HeLa cytoplasmic (CE), nuclear (NE), and DNA-bound cellular extracts were incubated with Aurora A in a kinase assay. As a control, a sample from nocodazole (Noc.)-treated HeLa cells was loaded. To verify the presence of Aurora A and MCM2, immunoblotting (IB) with anti-Aurora A and MCM2 antibodies was performed and also with anti-phosphohistone H3 Ser¹⁰ antibody to verify the quality of the DNA-bound extract. B, p160 signal appears only in the 1 m NaCl elution fraction of HeLa nuclear extracts. After fractionation and elution with different salt concentrations, unbound (UB) or 0.1, 0.3, 0.5, or 1 m NaCl elution fractions were incubated with Aurora A kinase, and the p160 signal was followed with anti-phospho-p160 antibody. As a control, unfractionated nuclear extract phosphorylated with Aurora A kinase was included. C, Coomassie Blue staining of cytoplasmic, nuclear, and 1 m NaCl (1 m) fraction. p160 protein is indicated. D, HeLa cells were transfected with two different RNAi oligonucleotides for BAF155 (1 and 2) or a pool of four different oligonucleotides for MYBBP1A. The luciferase gene RNAi-oligo (Luc) was used as a negative control. Cells were lysed after 72 h. Nocodazole treatment was performed for the last 18 h, and then cells were harvested and subjected to mitotic shake-off before total lysis. Immunoblots (IB) with anti-MYBBP1A, BAF155, MCM2, Aurora A, Aurora B, α-tubulin (loading control), and anti-phospho-p160 antibodies are shown. E, anti-phospho-p160 detects overexpressed MYBBP1A protein. HeLa cells were transfected with a FLAG-tagged version of MYBBP1A or the empty vector, and lysates were loaded and analyzed with anti-phospho-p160, anti-FLAG, or anti-α-tubulin (loading control) antibodies. F and G, the anti-phospho-p160 specifically detects phosphorylated MYBBP1A. Empty vector or FLAG-MYBBP1A-transfected HeLa cells were treated with 10 nm calyculin A for 1 h before lysis, and samples were probed against anti-phospho-p160, FLAG, Aurora A, Aurora B, or phosphohistone H3 Ser¹⁰ antibodies. F, FLAG-MYBBP1A-transfected HeLa cell lysates were treated with 400 units of λ-phosphatase for 1 h at 30 °C and, following SDS-PAGE and Western blotting, were probed with anti-phospho-p160 or anti-FLAG antibodies. Ponceau S staining shows the presence of λ-phosphatase.