CAS disrupts the interaction between Vpr and NPI-1.

(A) Twenty-five pmol of purified recombinant RanQ69L and CAS were resolved by 10% SDS-PAGE and stained with CBB. (B) Glutathione-Sepharose beads coupled with the GST-Impα isoforms, Rch1, Qip1 and NPI-1 (each 25 pmol) or GST (25 pmol), were incubated with mRFP-Vpr, Q69LRanGTP (25 pmol) and/or CAS protein (5 and 50 pmol, respectively). The bound fractions of mRFP-Vpr and mRFP were analyzed by immunoblotting with anti-Flag M2 MAb. (C) The immunoblots of mRFP-Vpr binding were analyzed by densitometry and each sample was normalized to the Impα isoforms without CAS protein. Each column and error bar represents the means ± SD of results from three experiments. The asterisk* represents a p-value of <0.0005. (D) Glutathione-Sepharose beads coupled with the NPI-1 (each 25 pmol) or GST (25 pmol), were incubated with mRFP-Vpr, Q69LRanGTP (25 pmol) and/or CAS protein (10 and 50 pmol, respectively). The bound fractions of mRFP-Vpr were analyzed by immunoblotting with anti-Flag M2 MAb. The bound fractions and 1/20 of the input of mRFP-Vpr were analyzed by immunoblotting with anti-Flag M2 MAb. The positions of mRFP-Vpr are indicated.

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