Fig. 9. Competition between ERK and pORF3 for binding Pyst1.

A, schematic representation showing the experimental strategy. Lysates from cells expressing HA-tagged ERK were mixed with pORF3, and the mixture was incubated with either GST-tagged Pyst1 or GST alone bound to glutathione-Sepharose beads. After washing, the amounts of ERK and pORF3 trapped on the beads were estimated. B, either control (lanes 2, 4, 7, and 8) or ORF3-expressing (lanes 3, 5, 9, and 10) in vitro transcription-translation (TnT) lysates were mixed with lysates from control (lanes 4, 5, 7, and 9) or HA-ERK-expressing (lanes 2, 3, 8, and 10) cells. The TnT reactions were labeled with [³⁵S]methionine. The ERK retained on beads was estimated by Western blotting (WB) with anti-HA tag antibodies (lanes 1-6); the retained pORF3 was estimated by autoradiography (lanes 7-10). Lane 1 shows molecular size markers (kilodaltons), and lane 6 shows HA-ERK expression in the cell lysates. Arrows indicate HA-ERK (lower band in the doublet) and the pORF3 monomer or dimer. C, lysates from control (lane 2) or HA-ERK-expressing (lane 3) cells were incubated with GST beads. After washing, the beads were Western blotted with anti-HA tag antibodies. Lane 1 shows GST loading on beads, while lanes 4 and 5 show direct Western blotting of control or HA-ERK cell lysates, respectively, with anti-HA antibodies. D, lysates from HA-ERK-expressing cells (lanes 2-6) were mixed with 6, 10, 15, or 20 μg of purified recombinant pORF3 (lanes 2-5) or 20 μg of BSA (lane 6). Lysates from control cells were mixed with 20 μg of either pORF3 (lane 7) or BSA (lane 8). Following incubation with beads and washing, the retained HA-ERK was estimated by Western blotting. Lane 1 shows molecular size markers (kilodaltons), and lane 9 shows HA-ERK expression in the cell lysates.