After selecting the best binding sequence of 12-amino acids peptides, 2 types of peptide would be synthesized, plain peptide and peptide conjugated with fluorescein isothiocyanate (FITC). For the synthesis of the latter, a spacer region (GGGSCK) would be added at the C-terminal end of the peptide and FITC was conjugated with the side chain of lysine. Finally, C-terminal amidation would be performed. The synthesis of plain and FITC-labelled anti PN peptides was ordered from Syn Peptide company (Shanghai, China). FITC-labelled anti-PN peptide tested binding affinity to non-denaturing cell lysate of transfected BCA cells and their mock transfected cells and rPN by dot blot analysis. Briefly, 12.5 μg of cell lysate or 500 ng of rPN in 1 μl was applied onto nitrocellulose membrane. The membrane was blocked with 5% BSA followed by peptide incubation at 4 °C overnight and the fluorescent signal was detected the next day using the G:BOX gel documentation system. The checking of anti-PN peptide binding to intact PN-transfected BCA cells was also performed with similar process as immunocytochemistry plus a step of cell membrane permeabilizing after fixation by incubated with 1% Triton X for 1 min at RT. The single staining step was done by incubation of the permeabilized cells with 2 μM FITC-labelled anti-PN peptide for 1 h at RT, washed and then nuclear stained with Hoechst 33258. The observation was viewed under confocal microscope using laser diode 488 nm.

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