The H7N1 NS1 protein binds the cellular proteins RIL and Src.

<p><b>a</b>. Pull-down analysis of the interaction of human RIL with the H7N1 F3 protein. 10 µg of recombinant his-tagged F3 NS1 was added to glutahione (GSH) sepharose beads in the presence (lanes 2–4) or in the absence (lanes 5–7) of 10 µg of recombinant human GST-tagged RIL. Beads were washed with PBS1×. Bound proteins were luted with 10 mM GSH, separated on SDS-PAGE and detected by western blot with anti-GST (RIL) or anti.his (NS1) antibodies. W, wash; E, elution; B, beads. Lane 1, control western blot with input proteins. <b>b</b>. As in panel a, but with the F4<sub>Δ225–230</sub> protein. <b>c</b>. Pull-down of recombinant ZO1. Cell lysates overexpressing human ZO1 have been incubated with NiNTA beads in the presence (lanes 2–4) or in the absence (lanes 5–7) of recombinant his-tagged F4Δ225–230 protein. W, wash with 10 mM imidazole; E, elution with 500 mM imidazole; B, beads pellet. <b>d</b>. Recombinant GST-tagged human Src kinase was incubated in the presence (lanes 2,3) or in the absence (lanes 4, 5) of recombinant his-tagged H7N1 F3 NS1 protein. Proteins were pulled down with his-tag affinity NiNTA-agarose beads. Anti-Src or anti-his antibodies were used for the detection of Src and NS1, respectively. Lane 1, input proteins. Lanes 2 and 4, proteins detected in the supernatant in the presence or absence of F3, respectively. Lanes 3 and 5, pulled down proteins in the presence or absence of F3, respectively. <b>e</b>. Recombinant untagged human Src was added to NiNTA beads in the absence (lanes 1, 4) or in the presence of recombinant his-tagged F3NS1 (lanes 2, 3; 5, 6) and in the absence (lanes 2, 5) or in the presence of recombinant human GST-tagged RIL (lanes 3, 6). Lanes 1–3: pulled down proteins. Lanes 4–6, supernatants. Proteins were revealed by immunoblot with anti-Src (upper panel), RIL (middle panel) or his (bottom panel) antibodies. <b>f</b>. HeLa cell extracts were incubated in the presence (lanes 2, 3) or in the absence (lanes 5, 6) of his-tagged F3 NS1. Proteins were pulled down with his-tag affinity NiNTA-agarose beads. Lanes 2, 5:, proteins detected in the supernatant in the presence or absence of F3, respectively. Lanes 3, 6: pulled down proteins in the presence or absence of F3, respectively. Lanes 1, 4: inputs. Antibodies specific for the phosphorylated Y418 (pY418-Src) or Y529 phosphorylated (pY-529-Src) Src residues were used for the detection of the active and inactive Src forms, respectively. Anti-his antibodies were used for the detection of NS1.</p>