Primary IRAK1 and TRAF6 antibodies were obtained from Cell Signaling Technology (Danvers, MA). GAPDH was used as a loading control (Santa Cruz Biotechnology, Dallas, TX). Western Blotting was performed as described previously (36). Briefly, cells were collected and lysed, and protein was separated using NuPAGE pre-cast gels (Life Technologies Corp., Carlsbad, CA). Gels were transferred via semi-dry electrotransfer to 0.45 μm nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA), and probed with designated primary antibodies. Blots were then probed with LI-COR IRDye secondary antibodies and imaged using LI-COR Odyssey CLX according to the manufacturer's instructions (LI-COR, Lincoln, Nebraska). Analysis and relative quantification of gel bands was carried out using ImageJ software (NIH, Bethesda, MD) (37).