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Prostatic acid phosphatase is expressed in peptidergic and nonpeptidergic nociceptive neurons of mice and rats.

Taylor-Blake B, Zylka MJ - PLoS ONE (2010)

Bottom Line: Previously, we found that TMPase was molecularly identical to Prostatic acid phosphatase (PAP) using mice.Although PAP is found in peptidergic and nonpeptidergic neurons of mice and rats, the percentage of PAP+ neurons that express these markers differs between species.Collectively, our studies highlight similarities and differences in PAP localization within nociceptive neurons of mice and rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, UNC Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina, USA.

ABSTRACT
Thiamine monophosphatase (TMPase, also known as Fluoride-resistant acid phosphatase or FRAP) is a classic histochemical marker of small- to medium-diameter dorsal root ganglia (DRG) neurons and has primarily been studied in the rat. Previously, we found that TMPase was molecularly identical to Prostatic acid phosphatase (PAP) using mice. In addition, PAP was expressed in a majority of nonpeptidergic, isolectin B4-binding (IB4+) nociceptive neurons and a subset of peptidergic, calcitonin gene-related peptide-containing (CGRP+) nociceptive neurons. At the time, we were unable to determine if PAP was present in rat DRG neurons because the antibody we used did not cross-react with PAP in rat tissues. In our present study, we generated a chicken polyclonal antibody against the secretory isoform of mouse PAP. This antibody detects mouse, rat and human PAP protein on western blots. Additionally, this antibody detects PAP in mouse and rat small- to medium-diameter DRG neurons and axon terminals in lamina II of spinal cord. In the rat, 92.5% of all PAP+ cells bind the nonpeptidergic marker IB4 and 31.8% of all PAP+ cells contain the peptidergic marker CGRP. Although PAP is found in peptidergic and nonpeptidergic neurons of mice and rats, the percentage of PAP+ neurons that express these markers differs between species. Moreover, PAP+ axon terminals in the rat partially overlap with Protein kinase Cgamma (PKCgamma+) interneurons in dorsal spinal cord whereas PAP+ axon terminals in the mouse terminate dorsal to PKCgamma+ interneurons. Collectively, our studies highlight similarities and differences in PAP localization within nociceptive neurons of mice and rats.

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Chicken antibody detects mouse, rat and human PAP on western blots.(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.
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pone-0008674-g001: Chicken antibody detects mouse, rat and human PAP on western blots.(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.

Mentions: To generate a polyclonal antibody that reliably detects PAP in mouse tissues, we immunized chickens with the secretory isoform of full-length recombinant mPAP protein, purified as described previously [18]. This resulted in a high-titer antibody that recognized secretory mPAP protein in enzyme-linked immunosorbent assays (data not shown) and on western blots (Figure 1A). This antibody also recognized secretory hPAP protein on western blots, although the signal intensity was lower (Figure 1A). Since similar amounts of mPAP and hPAP protein were loaded, confirmed by staining a duplicate gel for total protein (Figure 1B), this suggested our chicken antibody had greater specificity for mPAP over hPAP protein. Lastly, this antibody recognized the transmembrane isoform of mouse PAP (mTM-PAP) and rat PAP (rTM-PAP) on western blots (Figure 1C).


Prostatic acid phosphatase is expressed in peptidergic and nonpeptidergic nociceptive neurons of mice and rats.

Taylor-Blake B, Zylka MJ - PLoS ONE (2010)

Chicken antibody detects mouse, rat and human PAP on western blots.(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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2800773&req=5

pone-0008674-g001: Chicken antibody detects mouse, rat and human PAP on western blots.(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.
Mentions: To generate a polyclonal antibody that reliably detects PAP in mouse tissues, we immunized chickens with the secretory isoform of full-length recombinant mPAP protein, purified as described previously [18]. This resulted in a high-titer antibody that recognized secretory mPAP protein in enzyme-linked immunosorbent assays (data not shown) and on western blots (Figure 1A). This antibody also recognized secretory hPAP protein on western blots, although the signal intensity was lower (Figure 1A). Since similar amounts of mPAP and hPAP protein were loaded, confirmed by staining a duplicate gel for total protein (Figure 1B), this suggested our chicken antibody had greater specificity for mPAP over hPAP protein. Lastly, this antibody recognized the transmembrane isoform of mouse PAP (mTM-PAP) and rat PAP (rTM-PAP) on western blots (Figure 1C).

Bottom Line: Previously, we found that TMPase was molecularly identical to Prostatic acid phosphatase (PAP) using mice.Although PAP is found in peptidergic and nonpeptidergic neurons of mice and rats, the percentage of PAP+ neurons that express these markers differs between species.Collectively, our studies highlight similarities and differences in PAP localization within nociceptive neurons of mice and rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, UNC Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina, USA.

ABSTRACT
Thiamine monophosphatase (TMPase, also known as Fluoride-resistant acid phosphatase or FRAP) is a classic histochemical marker of small- to medium-diameter dorsal root ganglia (DRG) neurons and has primarily been studied in the rat. Previously, we found that TMPase was molecularly identical to Prostatic acid phosphatase (PAP) using mice. In addition, PAP was expressed in a majority of nonpeptidergic, isolectin B4-binding (IB4+) nociceptive neurons and a subset of peptidergic, calcitonin gene-related peptide-containing (CGRP+) nociceptive neurons. At the time, we were unable to determine if PAP was present in rat DRG neurons because the antibody we used did not cross-react with PAP in rat tissues. In our present study, we generated a chicken polyclonal antibody against the secretory isoform of mouse PAP. This antibody detects mouse, rat and human PAP protein on western blots. Additionally, this antibody detects PAP in mouse and rat small- to medium-diameter DRG neurons and axon terminals in lamina II of spinal cord. In the rat, 92.5% of all PAP+ cells bind the nonpeptidergic marker IB4 and 31.8% of all PAP+ cells contain the peptidergic marker CGRP. Although PAP is found in peptidergic and nonpeptidergic neurons of mice and rats, the percentage of PAP+ neurons that express these markers differs between species. Moreover, PAP+ axon terminals in the rat partially overlap with Protein kinase Cgamma (PKCgamma+) interneurons in dorsal spinal cord whereas PAP+ axon terminals in the mouse terminate dorsal to PKCgamma+ interneurons. Collectively, our studies highlight similarities and differences in PAP localization within nociceptive neurons of mice and rats.

Show MeSH