FIG. 2.
PAT1 specifically interacts with the carboxyl-terminal 68 amino acids of Us11. (A) 35S-labeled PAT1 was incubated with purified GST fusion proteins containing the C-terminal domain of Us11 (Us11Δ1-87), the N-terminal domain of Us11 (Us11Δ88-155), or GST. After the complexes were isolated on glutathione-agarose beads, the proteins were separated by SDS-PAGE, and the fixed dried gel was exposed to Kodak film. The major translated product (input) corresponds to full-length PAT1. PAT1 specifically forms a complex with the C-terminal 87 amino acids of Us11. (B) GST fusion proteins were incubated with lysates prepared from 293T cells transfected with plasmids expressing FLAG-tagged, full-length PAT1. GST-containing complexes were collected on glutathione agarose beads, fractionated by electrophoresis in SDS-polyacrylamide gels, and transferred to a PVDF membrane. The membrane was probed with anti-FLAG antibody, followed by a horseradish peroxidase- conjugated secondary antibody, and PAT1 was visualized via enhanced chemiluminescence. (C) GST and the two GST-Us11 fusion proteins (GST-Us11Δ1-87 and GST-Δ88-155) were subjected to electrophoresis in SDS-polyacrylamide gels, blotted onto nitrocellulose membranes, renatured, and then probed with in vitro-translated [35S]methionine-labeled PAT1. The filter was washed and subsequently exposed in a phosphorimager cassette.