-synthesized capsaicin in protoplasts treated with or without anti-Pun1 antibodies. (A) Strategy for the cell-free assay of capsaicin synthesis. Protoplasts prepared from placental tissues of pungent peppers were opened in a hypotonic solution, including vanillylamine as a substrate, thus releasing enzymes immediately converted the exogenously added substrate to capsaicin using the endogenous 8-methyl-6-nonenoyl-CoA. Anti-Pun1 antibodies would inhibit this reaction by binding to the Pun1 protein. (B) Inhibition of capsaicin synthesis by anti-Pun1 antibodies. In the cell-free assay using protoplasts, de novo-synthesized capsaicin was analyzed in HPLC and quantified using a capsaicin standard calibration curve. The graph shows the data as fold change compared with the control [means ± SD relative to a control reaction treated with normal serum (set to 1.0) derived from four separate experiments]. The reduction in capsaicin synthesis in protoplasts with anti-Pun1 antibodies was statistically significant compared with control protoplasts using Student’s t-test. Asterisks indicate a significant difference at 0.05. A representative HPLC chromatogram is shown in Additional file 3: Figure S3.