FIG. 3.

Interaction of HBx and XIP in vitro. (a) SDS-PAGE analysis of IVT [³⁵S]methionine-HBx binding to GST-XIP. Lane 1, input IVT [³⁵S]methionine-labeled HBx; lanes 2 to 4, labeled HBx bound to Sepharose beads (lane 2), to Sepharose-GST beads (lane 3), and to GST-XIP fusion protein (lane 4). (b) SDS-PAGE analysis of immunoprecipitated IVT [³⁵S]methionine-labeled HBx and XIPF. Lane 1, XIPF immunoprecipitated by anti-FLAG M2 MAb (αF); lane 2, HBx immunoprecipitated by an anti-HBx MAb (αX) (19a). In lane 3, the αF MAb immunoprecipitated the HBx-XIPF complex, whereas in lane 4, the αX MAb did not. The αF MAb does not immunoprecipitate IVT [³⁵S]methionine-HBx, and positioning of the FLAG epitope at the amino terminus of XIP also did not permit immunoprecipitation of the HBx-FXIP complex (data not shown). We have been unable to prepare high-affinity antibodies against the small hydrophobic XIP.