KISS1 is secreted and processed into kisspeptins (KP) in multiple cell lines.

<p>A. (<i>Top panel</i>) Conditioned media collected from cultures of C8161.9, MelJuSo, MDA-MB-435, HeLa, COS7 and HEK293 cell lines which exogenously express KISS1. KP were immunoprecipitated with a monoclonal antibody detecting KISS1 (6a4.27), separated by Tris-tricine SDS-PAGE and then immunoblotted with anti-KISS1 antibody (1∶1000). (<i>Bottom panel</i>) Full-length KISS1 was detected in whole cell lysates using anti-KISS1 antibody as above (Mr∼15.9 kDa; Note: lower molecular mass bands were not detected from whole cell lysates as previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone.0084958-Nash2" target="_blank">[15]</a>). B. KISS1 processing into KP can be detected using immunoprecipitated KISS1/KP separated by Tris-tricine SDS-PAGE gel followed by Coomassie dye staining. KP bands were excised and analyzed by ESI-MS/MS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone.0084958.s001" target="_blank">Figure S1</a>]. KP sizes (approximate kDa) are predicted based upon the predicted PC cleavage sites in nascent KISS1 (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone-0084958-g002" target="_blank">Figure 2A</a>). KP sizes and sequence were confirmed by ESI-MS/MS and are shown schematically in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084958#pone.0084958.s001" target="_blank">Figure S1</a>.