Figure 3

(Top to Bottom) a: blot result of positive control B-Actin; b, c, d, and e are blot results of Core, E1, NS4A, NS4B respectively developed by AP conjugated Anti mouse with NBT/BCIP substrate (sigma). Cells were lysed and protein was extracted after 72 hrs after transfection for single stable clone after 3 weeks About 80-100 μg of total protein were loaded into each well on 12.5% SDS-PAGE and electrophoretically blotted onto a Hybond-C extra nitrocellulose membrane semi-dry blotting apparatus. The membrane was blocked for 1 hour with a 5% milk solution in Phosphate Buffered Saline-0.05% Tween (PBS-T), washed three times with 50 ml of PBS-T. A mixture of primary antibodies for structural genes core (sc-57800), E1 (sc-65459) and non structural gene NS4A (sc-52415), NS4B (sc-65457) was added at a concentration of 1:500-1:800 in 5 ml of PBS-T. After incubating at room temperature for 1 hour, the membrane was washed 3 times with PBS-T. A secondary antibody, rabbit anti-mouse IgG, conjugated to alkaline phosphatase was added at a dilution of 1/1000 in PBS-T, incubated at room temperature for one hour. The membrane was washed for three times with PBS-T. Substrate tablet (NBT/BCIP) was dissolved in 1XPBS and blot was incubated for 15-30 min.