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. 2011 Sep;85(17):9090–9102. doi: 10.1128/JVI.00666-11

Fig. 8.

Fig. 8.

Binding of GAPDH (Tdh2p) to TBSV p33 and p92 proteins. (A) Schematic representation of viral proteins and their derivatives used in a binding assay. The various domains include the following: TMD, the transmembrane domain; RPR,the arginine-proline-rich RNA-binding domain; P; phosphorylated serine and threonine; and the S1 and S2 subdomains involved in p33:p33/p92 interaction. The two RNA binding regions in p92 are shown with gray boxes. (B) In the top panel, a Western blot affinity-binding (pull-down) assay was performed to detect interaction between 6×His/Flag-Tdh2p and the MBP-tagged viral proteins. The MBP-tagged viral proteins and MBP produced in E. coli were immobilized on amylose-affinity columns. The yeast lysate containing 6×His-tagged Tdh2p was then passed through amylose-affinity columns with immobilized MBP-tagged proteins. The affinity-bound proteins were specifically eluted with maltose from the columns. The eluted 6×His-tagged Tdh2p was analyzed by Western blotting with anti-6×His antibody. For the bottom panel, the amounts of MBP-tagged proteins bound to the amylose columns were analyzed by Coomassie blue staining of SDS-PAGE gels. (C) Pulldown assay to detect interaction between 6×His-RBD1+2 (the N-terminal RNA-binding domains of Tdh2p) and the MBP-tagged p92 viral protein. The MBP-tagged viral proteins and MBP were immobilized on amylose affinity columns as described for panel B. The yeast lysate with 6×His-tagged RBD1+2 was then passed through the amylose affinity columns with immobilized MBP-tagged proteins. The eluted 6×His-tagged RBD1+2 was analyzed by Western blotting with anti-6×His antibody. Total sample represents the crude yeast cell lysate. (D) A split-ubiquitin assay was used to test binding between p33/p92 and Tdh2p in yeast. The bait p33 or p92 was coexpressed with the indicated prey proteins. Ssa1p (HSP70 chaperone) and the empty prey vector (NubG) were used as positive and negative controls, respectively. (E) Pulldown assay to detect interaction between 6×His-Tdh2p and the TCV MBP-p88 and MBP-p88C proteins. See further details in panel B.