Skip to main content
. 2006 Oct 11;80(24):12041–12048. doi: 10.1128/JVI.01425-06

FIG. 1.

FIG. 1.

WDSV rv-cyclin blocks pulldown of proteins by GST-VP16. A. Model of rv-cyclin illustrating regions characterized by sequence analysis (Cyclin Box) or by functional assays (nuclear localization and AD). Numbers represent amino acid positions. Valine at position 260 within the defined AD is indicated (V260). B. Western blot of pulldown by GST-VP16 AD from 75 μg HeLa nuclear extract (nb [no block]) and from matching extracts preincubated with increasing twofold concentrations (20 to 160 μg) of recombinant His-rv-cyclin (aa 1 to 297) or carboxy-truncated His-rv-cyclin (aa 1 to 219). Blots were probed consecutively with antibodies reactive to p300 (αp300), Sur-2 (αSur-2), and GST (αGST), which recognizes the input GST-VP16 AD fusion protein. A portion of the unbound fraction, representing 20% of total input, was subsequently precipitated and run separately to assess His-tagged proteins with anti-poly-His antibody (αHis; bottom panels). C. Pulldowns by GST-VP16N (aa 413 to 452) and GST-VP16C (aa 453 to 490) from 75 μg HeLa nuclear extract (nb) and from matching extracts preincubated with 20 μg wild-type (wt) or mutated (V260F and V260S) His-rv-cyclin carboxyl region (aa 219 to 297). Blots were probed consecutively with antibodies reactive to CBP (αCBP), p300 (αp300), Sur-2 (αSur-2), and GST (αGST), which recognizes the input GST-VP16N and GST-VP16C fusion proteins. The antibodies used for CBP and p300 are specific for these proteins and do not cross-react.