Fig 1: NS1-70 directly interacts with Ku70, HSPA8, and HSPA1b but not Ku80. (A) NS1-70Flag-His purification. NS1-70Flag-His was purified as described in Materials and Methods. The NS1-70Flag-His protein eluted from peaked fractions of the Sephadex G75 column was run on an SDS–10% PAGE gel and then stained with Coomassie brilliant blue dye. (B) Ku70Strep purification. Ku70Strep protein was expressed in BL21(DE3) component cells and purified with Strep-Tactin superflow. The purified Ku70Strep protein was analyzed on an SDS–10% PAGE gel and stained with Coomassie brilliant blue dye. Purified proteins were denoted by asterisks. (C to G) In vitro pulldown assay. Two micrograms of purified NS1-70Flag-His was used as bait to pull down 2 µg prey proteins, purified Ku70Strep (C), purified Ku70/Ku80 heterodimer (D), purified Ku80GST (E), purified HSPA8 (F), or HSPA1b (G) using anti-Flag affinity resin. As a negative control, 2 µg prey protein alone (minus bait) was incubated with anti-Flag resin in 400 µl of equilibrium buffer (lane 4 in panels C, D, E, F, and G). The proteins pulled down were analyzed by Western blotting using anti-Ku70 (C and D), anti-GST (E), anti-HSPA8 (F), or anti-HSPA1b (G) and using anti-Flag for NS1-70Flag-His (C to G); ~0.3 µg of the bait and prey proteins was loaded as inputs. In experiments for panels C and D, we first used 3 U of Benzonase to treat both the bait and prey protein at room temperature for 15 min to remove any DNA/RNA contaminations that may mediate the interaction.