Anti mbp

Aggrecan from bovine articular cartilage (cat no. A1960), and poly-L-ornithine hydrobromide (Mw 30,000–70,000; cat no. P3655) were purchased from Sigma-Aldrich. Recombinant human pleiotrophin produced in yeast was described previously [37 (link), 41 (link), 60 (link), 61 (link)]. Anti-PTPRZ-S, rabbit polyclonal antibodies against the extracellular region of PTPRZ [62 (link)], and rabbit polyclonal antibodies against phosphorylated Tyr-1105 of p190 RhoGAP [43 (link)] were described previously. The following are the sources of commercially available regents and antibodies used in the present study: Anti-aggrecan (clone Cat-315; cat no. MAB1581, Millipore), anti-MBP (cat no. sc-13914, Santa Cruz Biotechnology), anti-NG2 proteoglycan (cat no. AB5320, Millipore), anti-phosphotyrosine (clone PY20; cat no. ab16389, Abcam), anti-p190 RhoGAP (cat no. 610150, BD Biosciences; and cat no. 12164, Cell Signaling Technology), anti-FYN (cat no. P2992, Sigma-Aldrich; and cat no. 4023, Cell Signaling Technology), anti-phosphorylated Tyr-416 of Src (cat no. 2101, Cell Signaling Technology), anti-phosphorylated Tyr-527 of Src (cat no. 2105, Cell Signaling Technology), anti-GFAP (cat no. Z0334, Dako), anti-OLIG2 (cat no. AB9610, Millipore), and anti-APP (cat no. ab15272, Abcam).

Corpus callosum tissues and cultured cells were used for Western blotting analysis, as detailedly described by our previous study (Li et al., 2013b (link)). Total proteins were extracted using a compartment protein extraction kit (Millipore) according to the manufacturer’s instructions. After electrophoresed on 10% SDS–polyacrylamide gels, proteins in the gels were transferred onto PVDF membranes (Millipore), which were incubated with following antibodies: anti-CNPase (1:1000, Abcam), anti-GAPDH (1:3000, Kangwei Biotechnology, Shanghai, China), anti-MBP (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-PDGFαR (1:500, Abcam). After extensive washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:10000, Kangwei Biotechnology) for 1 h at room temperature and developed using an enhanced chemiluminescence western blotting detection kit (Kangwei Biotechnology). All band signals were quantified using ImageJ (NIH), and the data acquired were normalized to GAPDH expression and further normalized to the control.

Bone marrow cell nuclear extracts and a recombinant protein solution were mixed 1:1 with 2X Laemmli buffer and subjected to SDS-PAGE in a 10% polyacrylamide gel. The Precision Plus Protein Kaleidoscope Standard (Bio-Rad) was loaded into neighboring lanes as a molecular-weight marker. After electrophoresis, the separated proteins were transferred to PVDF membranes. The proteins were probed with primary anti-GATA2 (dilution ratio: 1:5000, Perseus Proteomics), anti-Lamin B (1:5000, Santa Cruz Biotechnology), anti-MBP (1:10,000, Santa Cruz Biotechnology), and anti-GST (1:5000, Santa Cruz Biotechnology) antibodies, followed by detection of the primary antibodies with horseradish peroxidase-conjugated secondary antibodies (1:5000-1:20,000, Invitrogen-Life Technologies). Signals were visualized on X-ray film using ECL-Prime Western blotting Detection Reagents (GE Healthcare) according to the manufacturer’s protocol. Signal values were quantified using ImageJ software.

Anti-MMP-12, anti-MMP-9, anti-GFAP, anti-MOG, anti-Iba1, anti-TNFα, anti-TNFR1, anti-TNFR2, anti-caspase-3, anti-cytochrome C, anti-AIF, and anti-MBP antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-NeuN antibody was obtained from Millipore (Billerica, MA). Anti-glial fibrillary acidic protein (GFAP) was obtained from Dakocytomation (Carpinteria, CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Novus Biologicals (Littleton, CO).

Recombinant proteins corresponding to the entire intracellular regions (ICRs) of PTPRZ1, PTPRA, and PTPRM were expressed using a baculovirus-silkworm expression system, and purified as described^{6 (link)}. The ICRs of PTPRG, PTPRS, and PTPRB and the catalytic domains of PTPN1 and PTPN6 were expressed as glutathione-S-transferase (GST) fusion proteins from each pGEX plasmid in Escherichia coli strain BL21 (ref. 6 (link)). GST fusion proteins were purified by glutathione affinity chromatography as described^{43 (link)}. Chondroitinase ABC (chABC) was purchased from Sigma-Aldrich (catalog #C3667). Anti-PTPRZ-S, rabbit polyclonal antibodies against the extracellular region of PTPRZ was described previously (ref. 44 (link)). The following are the specificities and sources of the commercially available antibodies used in the present study: Anti-RPTPβ (a monoclonal antibody against the intracellular domain of PTPRZ1 receptors, #610179, BD Biosciences), anti-SOX2 (#ab97959, Abcam), anti-POU3F2 (#12137, Cell Signaling), anti-OLIG2 (#AB9610, Millipore), anti-SALL2 (#12679–1-AP, Proteintech Group), anti-GAPDH (#ab9482, Abcam) anti-phosphotyrosine (PY20; #ab16389, Abcam), and anti-pY118-paxillin (#2541, Cell Signaling), mouse anti-paxillin antibody (#610569, BD Bioscience), anti-MBP (#sc-13914, Santa Cruz Biotechnology), and anti-NG2 proteoglycan (#AB5320, Millipore).

Brain samples were harvested and kept in 4% paraformaldehyde for 24 h and immersed in 30% sucrose for 3-4 days at 4°C. After cryoprotection in 30% sucrose, the brains were rapidly frozen in isopentane and stored at −80°C. Twenty micrometer cryostat sections at the level of the thalamus (bregma −3.3 mm) according to a stereotaxic atlas [15 ] were processed for immunohistochemistry. Immunohistochemistry analyses were performed with the UltraVision Quanto Detection System HRP DAB kit (Thermo Fisher Scientific, USA). The primary antibodies utilized were rabbit anti-GFAP antibody (glial fibrillary acidic protein, Santa Cruz Biotechnology, USA, 1 : 200 dilution), anti-VEGF (vascular endothelial growth factor, Millipore Corporation, USA, 1 : 400), and anti-MBP (myelin basic protein, Santa Cruz Biotechnology, USA, 1 : 200). The secondary antibodies utilized were biotinylated goat anti-mouse IgG and goat anti-rabbit IgG (Thermo Fisher Scientific, USA, 1 : 400 dilution). The results were observed using the Leica vertical microscope.