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. 2004 Sep;24(17):7419–7434. doi: 10.1128/MCB.24.17.7419-7434.2004

FIG. 7.

FIG. 7.

Spp2/p58 interacts with Orp2, and the interaction requires Spb70. Wild-type cells containing orp2+-three-FLAG either with or without spp2+-three-HA and mutant spb70-U12 containing orp2+-three-FLAG and spp2+-three-HA were cultured in EMM to mid-log phase at 25°C and then shifted to 36°C for 3.5 h. Whole-cell extracts (W.C.E.) were prepared from each strain. Presence or absence of HA-tagged Spp2/p58 and FLAG-tagged Orp2 in whole-cell extracts was shown by Western blotting (W.C.E. panel). Spp2/p58 proteins were immunoprecipitated from each strain with anti-HA antibody, and the immunoprecipitates were Western blotted with anti-HA for Spp2/p58 and anti-FLAG for Orp2 (I.P. Spp2 panel). In whole-cell extracts, Orp2-FLAG protein was detected in whole-cell extracts from spb70+ strains either with or without spp2+-three-HA (lanes 1 and 2) and from mutant spb70-U12 (lane 3), while Spp2-HA was not detected in whole-cell extracts from spb70+ cells without spp2+-three-HA (lane 4) and only detected in whole-cell extracts from spb70+ and spb70-U12 strains containing spp2+-three-HA (lanes 5 and 6). In the anti-HA immunoprecipitates (I.P. Spp2 panel), Orp2-FLAG coprecipitated with Spp2/p58 from whole-cell extracts in the spb70+ strain containing spp2+-three-HA (Lane 8) but not from mutant spb70-U12 containing spp2+-three-HA (lane 9). Anti-HA immunoprecipitates from whole-cell extracts of the spb70+ strain without spp2+-three-HA had neither detectable Orp2-FLAG nor Spp2/p58-HA (lanes 7 and 10), and Spp2-HA was detected in immunoprecipitates from whole-cell extracts of spb70+ and spb70-U12 strains containing spp2+-three-HA (lanes 11 and 12).