Western Blot Analysis of Immune Signaling

Cell lysates were prepared as previously described [21] (link) following stimulation as described above. Samples were separated using SDS-PAGE on Mini-protean TGX Any kD gels (Bio-rad) and transferred onto a nitrocellulose membrane using a wet transfer system. Membranes were blocked, washed, and proteins were analyzed by immunoblotting with standard methods using antibodies specific to IRF-7, IRF-3 (both from Cell Signaling Technology) and GAPDH (Abcam). Secondary antibodies conjugated to HRP were obtained from Jackson ImmunoResearch and immunoreactive bands were detected with the Immuno-Star HRP Substrate kit (Bio-Rad). For time courses, cells were stimulated as described above and lysed directly in Laemmli buffer (Bio-rad) and equal volumes were assayed as with other western blots using antibodies specific to STAT1 and pSTAT1Y701 (Abcam). Densitometry was performed using ImageJ (NIH), and proteins of interest were normalized to a reference protein (GAPDH).