Chemocam 6.0 ecl system

Cells were washed once with PBS and lysed in Western blot lysis buffer (1% Triton X-100). After sonification and denaturation at 95°C, the protein concentration was measured by Bradford assay. Cell lysates were mixed with Bradford reagent (1:5) and absorbance was measured at 595 nm. For each sample, 10 μg of total lysate was resolved by electrophoresis in a 10% or 15% sodium dodecyl sulfate-polyacrylamide gel for NS3 or GFP, respectively. Proteins were transferred to a polyvinylidene fluoride membrane overnight. Membranes were blocked in skim milk (5%) in PBS-T (0.2%) for 1 h at RT. After washing thrice with PBS-T (0.2%) for 15 min, membranes were incubated with primary antibody for 1 h at RT. The membranes were washed thrice and horseradish peroxidase (HRP)-conjugated secondary antibody was added. After incubation for 1 h at RT, the membrane was washed thrice and the bound antibodies were detected using enhanced chemiluminescence solution (Perkin Elmer, Waltham, MA, USA). Images were acquired using the ChemoCam 6.0 ECL system (INTAS Science Imaging, Goettingen, Germany).

Cells were lysed using protein sample buffer, followed by sonication and denaturation at 95°C for 10 minutes. 10 μg of total protein from each sample was resolved by SDS-PAGE and transferred to nitrocellulose. Membranes were blocked with PBS-T (PBS - pH 7.4, containing 0.1% Tween-20) containing 5% skim milk for 2 h at room temperature, followed by incubation with anti-sera containing primary antibodies overnight at 4°C (for list of primary and secondary antibodies used see SI Table 5 and 6). Membranes were washed and incubated for 1 h with the HRP-conjugated secondary antibodies. Membranes were imaged using the ChemoCam 6.0 ECL system (INTAS Science Imaging, Goettingen, Germany). Images were cropped and analyzed using Fiji software (National Institute of Health).