Rep8 Is a Tissue-Specific p97 Cofactor Figure 1. Rep8 interacts with p97 via the UBX domain. (A) Yeast two-hybrid analyses of the p97-Rep8 interaction using the HIS3 reporter gene. Co-transformation of bait p97 with prey Rep8 supported cell growth under conditions selecting for the interaction (in the absence of histidine and presence of 25 mM 3-aminotriazol (3AT); right panel). The known p97-p47 interaction served as a control. (B) Schematic diagram of the Rep8 domain organization and the various truncations used in the precipitation experiments. (C) Purified p97 was incubated with the indicated, immobilized GST fusion proteins before analysis of bound proteins by SDS-PAGE and blotting using antibodies specific for p97 (top panel) or GST (lower panel). doi:10.1371/journal.pone.0025061.g001 HeLa cell components separated by differential centrifugation. As a control, we analyzed the fractions for the peripheral membrane protein p97 and the transmembrane protein calnexin (Fig. 3A). The association of these proteins with membranes were as expected, indicating that the membrane fractionation was successful. We found that all Rep8 was associated with the high- speed pellet and was released when the membranes were treated with detergent but not by washing with sodium chloride or sodium carbonate (Fig. 3A), indicating that Rep8 is indeed, as predicted, a transmembrane protein. We also analyzed the localization of Rep8 by fluorescence microscopy. Since our antibodies to Rep8 were not suitable for Figure 2. Rep8 interacts with the p97 N-domain. (A) Schematic diagram of the p97 domain organization and the various truncations used in the precipitation experiments. (B) Purified 6His-tagged p97 and p97 truncations were incubated with immobilized GST or GST-tagged Rep8 before analysis of the bound proteins by SDS-PAGE and blotting using antibodies specific for the 6His-tagged p97 proteins (top panel) or GST (lower panel). The p97 input has been included for comparison. (C) Endogenous p97 was immunoprecipitated from HeLa cell lysates. The precipitated material was analyzed by blotting using antibodies to p97 and Rep8. (D) Endogenous p97 was immunoprecipitated from rat testis homogenate. The precipitated material was analyzed by blotting using antibodies to p97 and Rep8. doi:10.1371/journal.pone.0025061.g002 PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e25061