FIG 4.

NP1 specifically interacts with CPSF6 in vitro. (A) NP1-His purification. NP1-His was purified as described in Materials and Methods. The NP1-His protein eluted from fractions 1 to 5 (lanes F1 to F5, respectively) was run on an SDS-PAGE gel (15% acrylamide) and then stained with Coomassie brilliant blue dye. (B) CPSF6-Flag purification. CPSF6-Flag protein was expressed in suspension-culture 293-ES cells by transfection and purified with anti-Flag beads. Four micrograms of the purified CPSF6-Flag protein was analyzed on an SDS-PAGE gel (10% acrylamide) and then stained with Coomassie brilliant blue dye. (C and D) In vitro pulldown assay. (C) Purified NP1-His and control MBP-His were used as the bait to pull down purified CPSF6-Flag using Ni-NTA agarose beads. The proteins pulled down by the Ni-NTA agarose beads were analyzed by Western blotting using anti-His (bottom) and anti-Flag (top). (D) Purified CPSF6-Flag was used to coat anti-Flag M2 affinity beads, which were used to pull down purified NP1-His and MBP-His (a negative control). The proteins pulled down by the anti-Flag M2 affinity beads were analyzed by Western blotting using anti-His (top) and anti-Flag (bottom). Lanes M, molecular mass markers.