Western Blot and ELISA Analysis of IFNβ

Cells were sonicated in radioimmunoprecipitation assay buffer (RIPA) or Laemmli buffer, and proteins were analyzed by Western blot, as described [28 (link)]. β-Actin level was also determined as a control. Western blot images were acquired using Kodak Digital Science Image Station 440CF. Monoclonal F/ARFP antibodies were generated against recombinant JFH1 F protein (J.-H. James Ou from University of Southern California). IFNβ concentrations in the cell culture medium were determined by ELISA with or without concentrating the samples, using Human IFNβ ELISA kit from Interferon Source, Inc. and using IFNβ as standards. Individual data points for ELISA are presented in S1 Table. For concentrating the samples, an Amicon Ultra-15 device (EMD Millipore) was used. Culture medium was pipetted into the Amicon Ultra-15 device and then centrifuged for 30 min at 3,000g (4°C). For immunofluorescence staining, samples were fixed, permeabilized, and incubated with primary antibodies, followed by incubation with fluorophore-conjugated secondary antibodies, and imaged by confocal laser scanning microscopy, as described [28 (link)]. Images were quantified by ImageJ available at http://rsbweb.nih.gov/ij/. Individual data points for ImageJ quantification are presented in S1 Table.