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. 2008 Mar 11;105(11):4352–4357. doi: 10.1073/pnas.0712276105

Fig. 2.

Fig. 2.

Mapping the binding domains of ZAP and the p72 RNA helicase. (A) Bacterially expressed GST or GST-p72 was immobilized onto glutathione-Sepharose 4B resin and incubated with the lysates of the cells expressing the indicated myc-tagged ZAP proteins in the presence of RNase A at 4°C for 2 h. The resins were washed and boiled in the sample loading buffer. (Upper) The proteins were resolved by SDS/PAGE and detected by Western blotting using the anti-myc antibody. (Lower) Schematic representations of the ZAP proteins are shown, and their binding activities are summarized. Input, total cell lysate; Ctrl, control cells. (B) The plasmid expressing the indicated myc-tagged ZAP protein and the plasmid expressing Flag-tagged p72 were transiently cotransfected into HEK293T cells. The cell lysates were immunoprecipitated with the anti-Flag antibody in the presence of RNase A and Western-blotted with the anti-myc antibody. Input, total cell lysate; Ctrl, control plasmid pCMVHF; EV, empty vector pcDNA4. (C) Bacterially expressed recombinant proteins of the indicated p72-truncation mutants were immobilized onto glutathioneSepharose 4B resin and incubated with the lysates of control cells or ZAP-expressing cells in the presence of RNase A at 4°C for 2 h. The resins were washed and boiled in the sample loading buffer. (Upper) The proteins were resolved by SDS/PAGE and detected by Western blotting using the anti-myc antibody. (Lower) Schematic representations of the p72 proteins are shown and their binding activities are summarized. Input, total cell lysate; C, control cells; Z, ZAP-expressing cells. The positions of the GST proteins are indicated by asterisks. (D) The plasmid expressing the indicated Flag-tagged p72 proteins were transiently transfected into ZAP-expressing cells or control cells. The cell lysates were immunoprecipitated with the anti-Flag antibody in the presence of RNase A and Western-blotted with the indicated antibodies. Input, total cell lysate; C, control cells; Z, ZAP-expressing cells. EV, empty vector. The positions of the p72 proteins are indicated by asterisks.