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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.

After treatment with desired concentrations of Bis-I, Gö6976, and Rottlerin, cells were harvested and washed in PBS. The extracts were prepared by resuspending cell pellets in lysis buffer (50mM Tris-HCI, pH 8.0, 150 mM NaCI, 1% Triton X-100, 5 mM EDTA), briefly sonicated and centrifuged at 15000xg for 15 min at 4°C. The protein concentrations were determined using BCA assay (Thermo). SDS-PAGE and immunoblotting were performed. Nitrocellulose membranes were probed with anti-PKCα, anti-PKCδ, anti-PKCη, anti-PKCγ, anti-PKCζ, anti-GAPDH (sc-208, sc-213, sc-215, sc-211, sc366126, sc-25778, Santa Cruz Biotechnology), anti-PKCβ, anti-PKCϵ, anti-claudin-1, −3, −4, and −7, anti-CD9, anti-CD82, Tspan-8 (Abcam), anti-caspase 3 and 9, anti-LC3A/B, Anti-Atg-5, anti-EpCAM, anti-E-cadherin, anti-vimentin (Cell Signaling). Immunoblotting signals were developed using chemiluminescence. All experiments were performed in triplicate.

Animals were transcardially perfused with 4% PFA in PBS. Spinal cord and brain tissues were post-fixed with 4% PFA in PBS at 4 °C overnight, cryoprotected in 30% sucrose in PBS, and then embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura Finetek, Torrance, CA, USA) for frozen sectioning. Coronal sections were cut at 30-μm thickness on a cryostat and mounted on Matsunami adhesive silane-coated glass slides (Matsunami Glass, Osaka, Japan).
For histology, sections were stained with cresyl violet (Nissl stain; Sigma). Brain lesion volume was estimated by measurement of the area of lost tissue in each of three to five sections spaced 0.5 mm apart. The total lesion volume was calculated as described previously.^{36 (link)}For immunohistochemistry, sections were permeabilized in PBS containing 0.1% X-100 and 0.5% BSA for 1 h at room temperature. Sections were then incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 2 h at room temperature. The primary antibodies used were as follows: mouse anti-NeuN (1 : 100; Merck, Cat# MAB377, RRID: AB_11210778), rabbit anti-PHD2 (1 : 200; Abcam), rabbit anti-PKCγ (1 : 100; Santa Cruz, Cat # sc-211, RRID: AB_632234). Alexa Fluor 488- and 588-conjugated goat anti-mouse IgG and goat anti-rabbit IgG antibodies (1 : 500; Invitrogen) were used as secondary antibodies.