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Validation of TAILS-predicted cleavage site by in vitro cleavage assay. (A) Schematic of a cytomegalovirus (CMV) promoter-driven mammalian expression construct containing 3×FLAG and 3×HA fused in-frame with the full-length candidate substrate. (B) Lysates from HeLa cells expressing the FLAG-HA-tagged wild-type or mutant candidate substrate were incubated with wild-type or mutant poliovirus 3Cpro and immunoblotted for FLAG. N, N-terminal cleavage product.
