Comparison of the distribution of proSAAS-derived peptides and NPY-expressing cells in the arcuate nucleus of the hypothalamus.

<p>Immunofluorescence was performed in mice expressing GFP under the NPY promoter, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104232#s2" target="_blank">Materials and Methods</a>. Left panels show the distribution of GFP, which is localized to the cytosol and reveals the cell bodies of NPY-positive neurons. Middle panels show immunofluorescence of the indicated proSAAS-derived peptide. Right panels show the merged images from the left and right panels. Cell nuclei were visualized with DAPI (blue color, right panels). Scale bar = 10 µm.</p

Effect of mutation of the two major furin cleavage sites on the localization of SAAS and PEN in AtT-20 cells.

<p>Wild type rat proSAAS was transiently expressed in AtT20 cells and peptides examined by immunofluorescence as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104232#s2" target="_blank">Materials and Methods</a> using antiserum to PEN (Cy3) and SAAS (Alexa 488 directly labeled). A: Wild type rat proSAAS. B: Rat proSAAS with the two major furin sites (RxRR) mutated to KxRR, which eliminates the furin consensus site. C. Quantification of colocalization as measured by Pearson's Coefficient. At least 30 cells were averaged per condition. Error bars represent standard error of the mean. *, p<0.05 using Student's t-test.</p

Schematic diagram of proSAAS and proSAAS-derived peptides.

<p>The major peptides derived from proSAAS are indicated, along with the enzymes involved in the cleavages. The amino acids within the cleavage site are shown (top row); residues in black are removed by the carboxypeptidase (CPD or CPE), and residues in purple remain on the C-terminus of the upstream peptide. The sites that are efficiently cleaved by furin/CPD are indicated by solid arrows; these cleavages generate big SAAS, an intermediate peptide representing GAV and the mid-portion of proSAAS, and PEN-LEN. Furin can also cleave PEN-LEN into PEN and big LEN (dashed arrow) but this cleavage does not go to completion in mouse brain. Within secretory vesicles, big SAAS is cleaved into KEP and little SAAS, the middle portion of proSAAS is cleaved into GAV and an un-named peptide, PEN-LEN is cleaved into PEN and big LEN, and big LEN is converted into little LEN and a 4-residue peptide LLPP. The conversion of big LEN into little LEN and LLPP is primarily catalyzed by PC1/3 and CPE. In addition to these major forms of the proSAAS-derived peptides, smaller forms of little SAAS, GAV, and PEN are present in mouse brain although the levels of these truncated peptides are generally lower than the levels of the peptides indicated in this figure.</p

Comparison of proSAAS-derived peptide immunofluorescence in the arcuate nucleus of the hypothalamus in wild type and proSAAS knock-out mice.

<p>Immunofluorescence was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104232#s2" target="_blank">Materials and Methods</a>, using a Cy3-labeled secondary antiserum (red). Cell nuclei were visualized with DAPI (blue). Antisera to SAAS, PEN-LEN, PEN, big LEN, and little LEN show a strong punctate staining pattern in wild type mouse hypothalamus (left panels) which is absent in the knock-out mouse brain (right panels).</p

Localization of proSAAS-derived peptides in AtT-20 cells.

<p>Immunofluorescence was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104232#s2" target="_blank">Materials and Methods</a>. A: Big LEN (Cy2) and PEN-LEN (Cy3). B: Big LEN (Alexa 488 directly labeled) and PEN (cy3). C: Big LEN (Alexa 488 directly labeled) and little LEN (Cy3). D: SAAS (Alexa 488 directly labeled) and big LEN (Cy3). E: SAAS (Alexa 488 directly labeled, left panel) and PEN (Cy3, middle panel). The right panel is a merge of the two images. F. SAAS (Alexa 488 directly labeled, left panel) and little LEN (Cy3, middle panel). Right panel is a merge. Scale bars in panels A, B, C, and D indicate 10 µm. Scale bars in panels E and F represent 1 µm.</p

Localization of proSAAS-derived peptides in the arcuate nucleus.

<p>Immunofluorescence was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104232#s2" target="_blank">Materials and Methods</a> using antibodies directly labeled with Alexa 488 (big LEN and SAAS), or with unlabeled primary antisera followed by Cy2- or Cy3-labeled anti-rabbit or anti-chicken secondary antibodies. A: PEN-LEN (in red, Cy3) and big LEN (in green, Cy2). B: PEN-LEN (Cy3) and SAAS (Cy2). C: PEN (Cy3) and big LEN (Alexa 488 directly labeled). D: PEN (Cy3) and SAAS (Alexa 488 directly labeled). E: Little LEN (Cy3) and big LEN (Alexa 488 directly labeled). F: Little LEN (Cy3) and SAAS (Alexa 488 directly labeled). G: SAAS (Cy3) and big LEN (Alexa 488 directly labeled). Scale bar = 10 µm.</p