The full-length human POMT1 cDNA (GenBank entry BC065268.1) was cloned into the pTNT vector (GenScript, Piscataway, NJ, USA) under the control of the SP6 promoter, and multiplied in Invitrogen subcloning efficiency DH5α competent cells (ThermoFisher, Waltham, MA, USA), according to the manufacturer’s instructions. 35S-methionine-labeled POMT1 was produced by in vitro transcription and translation of the purified plasmids using the TNT Coupled Reticulocyte Lysate System according to the manufacturer’s instructions (Promega, Madison, WI, USA) in the presence of 35S-methionine (Perkin Elmer, Waltham, MA, USA). Unincorporated 35S-methionine was removed by gel chromatography on NAP-5 columns (GE Healthcare, Chicago, IL, USA). Plasma samples were analyzed for POMT1 (auto)-antibodies by RIA, as described for GAD autoantibodies21. Briefly, 2 µl of plasma was incubated overnight at 4 °C in 96-well plates with 20,000 cpm of 35S-methionine-labeled POMT1, diluted in 50 µl of TBST buffer (50 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.4). Antibody–antigen complexes were precipitated with protein A-Sepharose, and unbound label was removed by washing several times with TBST buffer (50 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.4). The bound radioactivity was measured with a liquid scintillation counter (1450 MicroBeta Trilux, Perkin Elmer Life Sciences, Turku, Finland), and the results were expressed in relative units (RU), based on a serial dilution in-house standard curve.

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