FIG. 8.
Attempted cross-linking of coexpressed PARN-FLAG and TTP-HA. Extracts were prepared from 293 cells expressing vector alone (BS+) or CMV-hTTP-tag (hTTP), CMV.hPARN.flag (hPARN), or both together. The extracts were then incubated without (−) or with (+) 3 mM MgCl2 for 20 min at 25°C as indicated. DSS (0.1 mM final concentration) was then added to some of the extracts (+, as indicated), and the extracts were rotated gently at 4°C for 45 min. The reactions were then stopped by the addition of Tris buffer (pH 8.0) to a final concentration of 0.1 M. Equivalent amounts of protein were then loaded onto three SDS-polyacrylamide gels, transferred to nitrocellulose, and blotted with antibodies to the FLAG epitope tag (A), PARN itself (B), or the HA epitope tag (C). Chemiluminescence autoradiography was then performed. The migration positions of molecular weight standards are indicated to the left of each blot. The brackets to the right of panels A and B indicate high-molecular-weight protein species that reacted with both the FLAG (A) and the PARN (B) antibodies. Immunoreactive PARN (A and B) and TTP (C) are also indicated. The faint immunoreactive species that occurred at Mr ∼100,000 in panel C may be TTP dimer. See the text for additional details.