Af4464

Tissue sections from 3 WT and 3
Entpd3
^-/- mice were stained immunohistochemically as previously reported (
Taylor-Blake & Zylka, 2010 (link)). Antibodies used were: polyclonal sheep anti-mouse ENTPD3/CD39L3 (skin, 1:75; DRG and spinal cord 1:400; AF4464, R&D Systems), monoclonal mouse anti-NeuN (1:250; MAB377, Millipore), polyclonal chicken anti-Prostatic acid phosphatase (1:4,000; PAP, Aves Labs), polyclonal rabbit anti-NF200 (1:800; N4142, Sigma), monoclonal mouse anti-NF200 (Clone RT97; MAB5262, Millipore), polyclonal rabbit anti-CGRP (1:150; BML-CA1134, Enzo Life Sciences), polyclonal sheep anti-CGRP (1:300; BML-CA1137, Enzo Life Sciences); polyclonal rabbit anti-PKCγ (1:800; sc-211, Santa Cruz), and polyclonal rat anti-PECAM1/CD31 (1:400; Clone MEC 13.3, 553370, BD Biosciences). IB4 conjugated with Alexa Fluor dyes and secondary antibodies conjugated with Alexa Fluor dyes were purchased from Invitrogen. DRAQ5 (Catalog # 4084) was purchased from Cell Signaling Technology. Stained sections were imaged on a Zeiss LSM 510 confocal microscope or a Zeiss LSM 710 confocal microscope.

Male WT and
Entpd3
^-/- (3 month-old; ~25 g; n=3 for each genotype) were decapitated, and the DRG and bladder tissue was collected and digested in modified RIPA buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% SDS, 0.5% deoxycholate) supplemented with protease inhibitors (Roche Complete Mini). Protein samples from the cell lysate (1 mg/ml) were analyzed by SDS-PAGE with 4–20% gradient Tris-Glycine polyacrylamide gels (BioRad). The protein samples were then transferred to a polyvinylidene difluoride membrane and probed with a sheep anti-ENTPD3 polyclonal antibody overnight at 4°C (0.2 μg/ml; AF4464, R&D Systems), followed by a rabbit anti-sheep horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature (0.16 μg/ml; #31480, Thermo Scientific).