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H.O. different preparations revealed that IVIg reacted with a 36-kDa membrane glycoprotein in the sciatic nerve. Subsequent analyses of peptide absorption identified the protein as a myelin protein in Rabbit polyclonal to HDAC6 the peripheral nervous system (PNS) known as large myelin protein zero (L-MPZ). Moreover, injected IVIg penetrated the demyelinating lesion, leading to deposition on L-MPZ in the myelin debris. These results indicate that IVIg may modulate PNS demyelination, possibly by binding to L-MPZ on myelin debris. Keywords: intravenous immunoglobulin (IVIg), demyelination, peripheral nerve, neuropathy, lysophosphatidylcholine (lysolecithin), large myelin protein zero (L-MPZ) 1.?Introduction The myelin structure Pitofenone Hydrochloride in the nervous system enables rapid propagation of nerve impulses along axons via saltatory conduction and the localization of axonal channels in specific regions.1C4) Patients with inflammatory demyelinating diseases of the peripheral nervous system (PNS) exhibit various clinical symptoms including symmetric muscle weakness with or without sensory disturbances ((Roche Diagnostics, Rotkreuz, Switzerland)/30 g of protein for 15 h at 30 and subjected to electrophoresis. 2.7.4. Electrophoresis and Western blotting. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed as previously described,16,17) with minor modifications. Briefly, the protein samples were mixed with sample buffer (62.5 mM Tris-HCl, pH 6.8, 5.0% sucrose, 2.0% SDS, 0.1 M DTT, containing with 0.002% bromophenol blue) and loaded onto 10.5% SDS-PAGE. Each sample was transferred to a PVDF membrane (pore size: 0.45 m; Merck Millipore, Tokyo, Japan). The membranes were incubated with 5.0% skim milk (Becton, Dickinson and Company, Franklin Lakes, NJ) in 20 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 0.1% Tween 20 (5.0% skim milk in T-TBS) for 60 min at room temperature to block non-specific reactions. Thereafter, membranes were incubated with IVIg (Glovenin? or Venilon?) as Pitofenone Hydrochloride the primary antibody (1:100C500) diluted in blocking buffer or T-TBS for 60 min, washed three times with T-TBS, and incubated with HRP-conjugated anti-human IgG antibodies in T-TBS for 30 min. After washing with T-TBS, immunoreactivity was detected using Amersham ECL (Cytiva, Tokyo, Japan). Immunoreactive signals were captured using an LAS-3000 (Fujifilm, Tokyo, Japan). The size markers were aligned with the positive bands. The analyzed membranes were stained with 0.1% amido black 10B to visualize loaded total protein. All antibodies used are listed in Table ?Table11. 2.8. L-MPZ peptide absorption test. We previously reported that the immunoreactivity of L-MPZ, a 36-kDa myelin protein, can be absorbed using serum IgG obtained from patients with CIDP.13) To determine the antigen involved in the present study, we examined the peptide absorption of the IVIg preparation using synthetic L-MPZ peptides. The peptides used for the absorption test were synthesized by GenScript (Piscataway, NJ) as follows: L-MPZ1C30 (RLAGRAGDRGLGVESAKGPKVMVIEMELRK), L-MPZ25C63 (EMELRKDEQSPELRPAVKSPSRTSLKNALKNMMGLNSDK), L-MPZ25C36 (EMELRKDEQSPE), L-MPZ32C43 (EQSPELRPAVKS), L-MPZ38C49 (RPAVKSPSRTSL), L-MPZ45C56 (SRTSLKNALKNM), L-MPZ52C63 (ALKNMMGLNSDK), and Pitofenone Hydrochloride a non-related (NR) scramble amino acid peptide (GLPGNEGPPGQK). Appropriately diluted IVIg solutions were incubated overnight at 4 with/without one of the synthetic peptides in TBS. The mixtures of IVIg and peptide were diluted 10-fold with 0.3% Pitofenone Hydrochloride skim milk in T-TBS. Anti-L-MPZ antibody from an immunized rabbit with L-MPZ37C56 peptide13) were also used as a positive control of 36-kDa protein. Sciatic nerve homogenates were loaded onto SDS-PAGE and transferred to a PVDF membrane. After blocking with 0.3% skim milk, the membranes were incubated with a mixture of IVIg and peptide, following which detection was performed using HRP-labeled anti-human IgG antibody. Membranes were incubated with anti-L-MPZ antibody (1:120,000), followed by HRP-labeled anti-rabbit antibody (1:10,000), to confirm that the 36-kDa protein was L-MPZ. The analyzed membranes were stained with amido black as described above. 2.9. Observation of L-MPZ and human IgG on myelin debris. After incubation in methanol at ?20 , the sections were blocked non-specific reaction with 10% NGS, and were incubated with rabbit anti-L-MPZ antibody (1:250). The sections were then visualized using Alexa Fluor 488-labeled goat anti-rabbit IgG (1:200) and Alexa Fluor 594-labeled goat anti-human IgG (1:200) antibodies. All antibodies used are listed in Table ?Table11. 2.10. Statistical analyses. All statistical analyses were performed using Prism 5 software (GraphPad Software, San Diego, CA). Data are expressed as the mean standard error (SE). Means were compared using Students t-test, Welchs t-test, or the Kruskal-Wallis test, followed by Dunns multiple comparison test. Differences were.