FIGURE 3.

Ligand binding by recombinant RIG-I protein and its derivatives. A, schematics of recombinant proteins expressed by baculovirus-infected insect cells. Two constructs expressed are full-length RIG-I and RIG-I with CARD domain deleted (R-HR). These constructs are expressed with and with out the GST tag that could be removed by treatment with thrombin. The site of cleavage is denoted with a black triangle. The constructs with GST are depicted with prefix g. The three domains of the RIG-I protein are shown in different colors, with key residue numbers shown. aa, amino acids. B, SDS-PAGE of the purified recombinant proteins. Each gel image shows the GST-tagged protein and the version in which the GST was removed and then purified away. M, molecular mass standards. C, results of UV cross-linking assay to examine ligand binding by the four versions of RIG-I protein. The top panel contains an image from an autoradiogram depicting the complexes of the RIG-I with the ligands named above the gel image. The bottom image contains the same gel that was stained with Coomassie Blue (C.B.) to reveal the location of RIG-I and its derivatives along with the internal negative control, BSA.