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. 2012 Nov 12;199(4):639–651. doi: 10.1083/jcb.201111042

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© 2012 Sivadas et al.

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Figure 7. — Two sites in RSP3 bind to the RIIa and the Dpy-30 domain. His-tagged RIIa from RSP7 (asterisk) or His-tagged Dpy-30 protein (arrowhead) were coexpressed with a GST-tagged RSP3 peptide (dot) in bacteria. The Ni-NTA pulldown from the extracts was analyzed by Coomassie-stained gel (A–D) or GST Western blots (E). (A) The positive control, the copurification of the RIIa domain, and RSP3_96–180 that contains the AH_R (Gaillard et al., 2001). (B and C) The copurification of His-tagged human Dpy-30 protein and GST-tagged RSP3_245–316 or RSP3_269–316 (dot). (D) The negative control. GST alone did not interact with Dpy-30 protein. (E) The smallest region that binds the Dpy-30 domain is aa 280–308 in RSP3. The interaction is perturbed by V₃₀₀P mutation. His-tagged full-length Dpy-30 protein or the Dpy-30 domain was only coexpressed with GST-tagged RSP3 peptides as indicated. The irregular patterns in the Ponceau-stained membranes for the demonstration of protein loads (middle and bottom) were from plastic wraps and staining solution. Pre, the bacterial extract; Post, the flow-through from Ni-NTA matrix; and E, one of the eluates that contained the most Dpy-30 (see Materials and methods). Dpy-30 often migrated as double bands because of the susceptibility of its C-terminal end to proteolysis.