Figure 1.

Association of PP1α with γ₁34.5 and MyD116. (A) Replicate HeLa cell cultures were harvested in lysis buffer containing 10 mM Hepes (pH 7.6), 250 mM NaCl, 10 mM MgCl₂, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, and 2 mM benzamidine 18 hr after mock infection or infection with 10 pfu of HSV-1(F) or R8300 per cell. After 30 min on wet ice and low speed centrifugation to remove nuclei, the supernatant fluids were precleared with GST beads and then reacted with GST phosphatase 1 bound beads at 4°C overnight. After extensive rinsing, the proteins bound to beads were solubilized by boiling in disruption buffer containing 50 mM Tris·HCl (pH 7.0), 5% 2-mercaptoethanol, 2% SDS, and 2.75% sucrose, electrophoretically separated on denaturing 12% polyacrylamide gels, and transferred to a nitrocellulose sheet, and the blot was probed with anti-γ₁34.5 serum (1). The positions of γ₁34.5 protein and of the chimeric protein γ₁34.5–MyD116 are shown. (B) An aliquot of GST–PP1α fusion protein bound to beads was reacted with 25 units of thrombin (Sigma) in PBS at room temperature. After 8 hr, the mixture was spun in a table top centrifuge and the supernatant fluid containing PP1α was then dialyzed against lysis buffer, reacted with GST, GST–MyD116_C, or GST–γ₁34.5_C bound to beads and processed as described in A. PP1 was detected with antiphosphatase 1α antibody (Upstate Biotechnology, Lake Placid, NY).