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Q-to-R mutations in Sec9 produce a substantial decrease in binding affinity. Sec9 proteins were added in increasing concentrations (0.01–10 μM) to the assembly reactions, incubated overnight, and immunoblotted with α-Sec9 antibody (left panel). The amount of Sec9 incorporated into the SNARE complex was quantitated and used to calculate EC50 values (right panel).
