Western Blot Analysis of Protein Targets

Cell lysates were prepared at the indicated time points postinfection in Laemmli sample buffer (Bio-Rad) supplemented with 355 mM 2-mercaptoethanol (BME). The samples were boiled for 5 min, separated on a Bolt 4 to 12% Bis-Tris gel (Invitrogen), and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% (wt/vol) nonfat dry milk in Tris-buffered saline (TBS) supplemented with 0.5% Tween 20, and proteins were detected by incubation with primary antibodies diluted in blocking buffer followed by incubation with secondary antibodies (raised in goat against the appropriate species) conjugated to horseradish peroxidase (HRP) and diluted in blocking buffer. HA was detected using an HRP-conjugated HA antibody (Roche 12013819001), V5 was detected with mouse anti-V5 tag monoclonal antibody (Invitrogen R960), SAG2A was detected using rabbit polyclonal anti-SAG2A antibodies (55 (link)), MAF1b was detected using rabbit polyclonal anti-MAF1b antibodies (27 (link)), GAPDH was detected using mouse monoclonal anti-GAPDH antibody 6C5 (Calbiochem), and biotinylated proteins were detected using streptavidin-HRP (Invitrogen S911). HRP was detected using an enhanced chemiluminescence (ECL) kit (Pierce). Silver-stained gels were generated using the Pierce silver stain kit (Thermo Scientific).