Figure 2.

The NSP2/GIGYF2-4EHP interaction involves multiple binding sites

(A) Schematic cartoon of the V5-tagged GIGYF2 fragments used in panel (B). 4EHP-BM: 4EHP-binding motif; GYF: glycine-tyrosine-phenylalanine domain; SAH: putative single alpha helix; polyQ: glutamine-rich stretches.

(B) Western blot showing the interaction between Flag-NSP2 and two regions in GIGYF2. Vectors expressing Flag-NSP2 and the indicated fragments of V5-tagged GIGYF2 were transiently transfected in HEK293T to perform Flag IP. Extracts were RNase A-treated. Empty vectors were used as negative controls (−). FL: Full-length GIGYF2.

(C) Ni-NTA pull-down assay showing the interaction between recombinant His₆-NSP2 and untagged GIGYF2^743−1,085. GST served as negative control. The starting material (Input) and bound (Ni-NTA pull-down) fractions were analyzed by SDS-PAGE followed by Coomassie blue staining.

(D) Western blot showing the interaction between Flag-NSP2 and V5-4EHP in a GIGYF2-independent manner. RNase A-treated extracts from cells expressing Flag-NSP2 along with V5-4EHP, WT or carrying the W95A substitution (Mut), were used for Flag IP. Inputs and bound fractions were analyzed by Western blotting using the indicated antibodies. Empty vectors served as negative controls (−).

(E) Ni-NTA pull-down assay showing the simultaneous interactions between recombinant His₆-NSP2 and both untagged 4EHP and GIGYF2^743−1,085. Incubations were performed with the indicated recombinant proteins. The starting material (Input) and bound (Ni-NTA pull-down) fractions were analyzed by SDS-PAGE followed by Coomassie blue staining.