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After primary antibody incubation, slices were then washed with PBS three times and incubated with secondary antibody donkey anti-mouse IgG (H + L) Alexa Fluor 488 (1:1000, Thermofisher Scientific, A-21202) in blocking solution for 90 min

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In PDB ID 1nu8, Diprotin A forms a tetrahedral intermediate, where the C atom of the carbonyl group of Pro2 in Diprotin A was found in a tetrahedral configuration (grey ball in Figure 5) covalently linked to Ser630