Fig. 2. In vitro binding of the three Hrp65 isoforms. Recombinant His-tagged Hrp65-1, -2 and -3 were purified on Ni-NTA-agarose and incubated in the presence of ³⁵S-methionine labeled Hrp65-1, -2 or -3 obtained by in vitro translation in rabbit reticulocyte lysate (lanes 7-15). As a negative control, Ni-NTA agarose beads without His-tagged proteins were incubated in the presence of each ³⁵S-labeled Hrp65 isoform (lanes 4-6). The bound proteins were eluted, resolved in a 10% SDS-PAGE gel and autoradiographed. ³⁵S-labeled Hrp65-1, -2 and -3 proteins were loaded as inputs (lanes 1-3). The multiple Hrp65 bands observed in each input lane (bracket) are due to partial degradation and to alternative translation initiation sites. The mobility of molecular mass standards is indicated on the right (kDa).