Ecl peroxidase detection system

KHYG-1 cells were harvested, washed with PBS, and lysed in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM KCl, 1.5 mM MgCl₂, 1% (w/v) Triton X-100, 1 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). After incubation on ice for 20 min, debris was removed by centrifugation at 2000 × g for 10 min at 4 °C. Supernatant was used as a loading sample. Proteins (30 μg) were subjected to SDS-poly-acrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Nonspecific binding was blocked by incubation of the membrane with PBS containing 0.1% (w/v) Tween-20 (PBS-T) and 5% (w/v) non-fat dried milk for 20 min at room temperature. Then membrane was incubated with primary antibodies overnight at 4 °C and with secondary antibodies for 45 min at room temperature. Immunoreactive protein bands were visualized using an ECL-peroxidase detection system (Amersham Biosciences, Piscataway, NJ, USA) and LAS-4000 (Fujifilm, Tokyo, Japan).

To detect JEV-E protein by western blot analysis, cells or media were harvested. Cells were lysed in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM KCl, 1.5 mM MgCl₂, 1% (v/v) Triton X-100, 1 mM PMSF, 10 μg/ml leupeptin and 10 μg/ml aprotinin). After incubation on ice for 20 min, debris was removed by centrifugation at 2,000 × g for 10 min at 4 °C. Supernatant and media were used as a loading sample. Proteins (30 μg) were subjected to SDS-poly-acrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Nonspecific binding was blocked by incubation of the membrane with PBS containing 0.1% (v/v) Tween-20 (PBS-T) and 5% (w/v) non-fat dried milk for 20 min at room temperature. Then membrane was incubated with primary antibodies overnight at 4 °C and with secondary antibodies for 45 min at room temperature. Immunoreactive protein bands were visualized using an ECL-peroxidase detection system (Amersham Biosciences, Piscataway, NJ, USA) and LAS-4000 (Fujifilm, Tokyo, Japan), and calculated with Image Gauge. JEV infection levels indicated as the ratio of JEV-E to actin.