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

a. 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. As in panel a, but with the F4Δ225–230 protein. c. 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. d. 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. e. 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. f. 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.