Chicken antibody detects mouse, rat and human PAP on western blots.

<p>(A) Western blot containing purified recombinant mPAP protein and pure hPAP protein probed with chicken (Ck) anti-PAP antibody. (B) Duplicate gel stained with GelCode blue to confirm that equivalent amounts of protein were loaded. (C) Western blot of cell lysates from untransfected HEK 293 cells and HEK 293 cells transfected with rTM-PAP or mTM-PAP.</p

Amino acid identity and similarity between the secretory isoforms of mouse, rat and human PAP.

<p>Calculated using BLASTP with GenBank accession #'s NP_062781.2 (mPAP), NP_064457.1 (rPAP) and NP_001090.2 (hPAP). The less conserved N- and C-terminal regions were not included in these alignments, resulting in higher percent identity values relative to a previous study with rPAP and hPAP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008674#pone.0008674-Roiko1" target="_blank">[20]</a>.</p

Chicken antibody detects PAP in mouse DRG neurons and dorsal spinal cord.

<p>(A–D) Sections from mouse L4-L6 DRG and (E–L) lumbar spinal cord were stained with chicken anti-PAP antibodies (red) and with antibodies against various sensory neuron markers and spinal interneuron marker PKCγ (blue, green). (D, H, L) Merged images. All images were acquired by confocal microscopy. Scale bar in (D) 50 µm, (H) 100 µm.</p

Chicken antibody detects PAP in rat DRG neurons and dorsal spinal cord.

<p>(A–D) Sections from rat L4-L6 DRG and (E–L) lumbar spinal cord were stained with chicken anti-PAP antibodies (red) and with antibodies against various sensory neuron markers and spinal interneuron marker PKCγ (blue, green). (D, H, L) Merged images. All images were acquired by confocal microscopy. Scale bar in (D) 50 µm, (H) 100 µm.</p

mPAP dephosphorylates purine nucleotides in a pH-dependent manner.

<p>Plot of initial velocity at the indicated concentrations of AMP, ADP and ATP at (A) pH 7.0 and (B) pH 5.6. Reactions (n = 3 per point) were stopped after 3 min. Inorganic phosphate was measured using malachite green. All data are presented as means±s.e.m. Error bars are obscured due to their small size.</p

CGRPα-GFP axons terminate in dorsal spinal cord.

<p>Sections of lumbar spinal cord from CGRPα-GFP^+/− mice were stained with antibodies to (A) GFP and (B) CGRP. (C) IB4-binding. (D) Merged image. Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bar in (D) is 100 µm.</p

CGRPα-GFP marks a small population of neurons in lamina II/III and motor neurons in spinal cord.

<p>Sections of lumbar spinal cord from CGRPα-GFP^+/− mice were stained with (A–F) antibodies to GFP (green) and PKCγ (red). IB4-binding (blue). Arrowheads point to GFP⁺ cells. (D–F) Single confocal scan image from box in (A) reveals a CGRPα-GFP⁺ neuron located in lamina II. (G–I) Motor neurons stained with antibodies to GFP (green) and CGRP (red). Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bar in (C and F) is 25 µm and in (I) is 100 µm.</p

CGRPα-GFP axons and cells in visceral tissues.

<p>Sections of (A–C) small intestine, (D–I), bladder and (J–L) thyroid from CGRPα-GFP^+/− mice were stained with antibodies to GFP (A, D, G, J) and the indicated markers. Nuclei in (A–C) were labeled with DRAQ5. (C-inset) Section of wild-type mouse small intestine stained for GFP (green) and DRAQ5 (blue). Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bars in (C) and (C-inset) inset are 100 µm and apply to (A–C). Scale bar in (L) is 50 µm and applies to (D–L).</p

Glabrous skin montage.

<p>Sections from the glabrous skin of the hindpaw from CGRPα-GFP+/? mice were stained with antibodies to (A) GFP and (B) PGP9.5. (C) Merged images were stained with the nuclear marker DRAQ5. Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bar in (C) is 50 μm.</p

TM-PAP dephosphorylates extracellular purine nucleotides in a pH-dependent manner.

<p>HEK 293 cells were transfected with expression vectors containing (A–F) mouse TM-PAP or (G–L) the fluorescent protein Venus as a control. Cells were then histochemically stained using AMP, ADP or ATP (each 6 mM) as substrate at pH 7.0 or pH 5.6. Cells were not permeabilized with detergent. Scale bar (bottom right panel), 50 µm for all panels.</p