Immunoblot and Line Blot

RGS8-His in 50 mM sodium phosphate pH 7.4, 8 M urea, 1,000 mM NaCl was incubated with NuPage lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific, Schwerte, Germany) containing 25 mmol/L dithiothreitol at 70°C for 10 minutes, followed by SDS-PAGE (NuPAGE, Thermo Fisher Scientific, Schwerte, Germany). Separated proteins were electrotransferred onto a nitrocellulose membrane by tank blotting with transfer buffer (Thermo Fisher Scientific) according to the manufacturer's instructions. The membranes were blocked with Universal Blot Buffer plus (EUROIMMUN, Germany) for 15 minutes and incubated with the patient or control sera (dilution 1:200) in Universal Blot Buffer plus for 3 hours.

Alternatively, purified RGS8-His was coated as a line onto nitrocellulose membrane, and strips were incubated with sera (1:100 dilution) in Sample buffer (EUROIMMUN, Germany) for 30 minutes. Serum incubation was followed by 3 washing steps with Wash buffer (EUROIMMUN, Germany), a second incubation for 30 minutes with alkaline phosphatase–labeled anti-human-IgG (1:10, EUROIMMUN, Germany), 3 washing steps, and staining with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate substrate (EUROIMMUN, Germany) for 10 minutes. After having dried, line strips were evaluated by the use of EUROLineScan software (EUROIMMUN, Germany). For the detection of anti-SOX1 reactivity, a SOX1 line blot (EUROIMMUN, Germany) was performed according to the manufacturer's instructions.