Figure 8. Blockade of autophagy impairs TLR-triggered TRAF6 ubiquitination and MAP3K7 activation. (A and B) A549 cells were transfected with control siRNA or siRNA specific for ATG5 (A) or ATG7 (B). Forty-eight hours later, cells were treated with LPS (10 μg/ml) or poly(I:C) (20 μg/ml) for 12 h or left untreated. Whole cell lysates were immunoprecipitated with anti-TRAF6 antibody and then immunoblotted with anti-K63 specific ubiquitin and anti-TRAF6 antibody. (C) A549 cells were transfected with control siRNA or siRNA specific for ATG7. Forty-eight hours later, cells were treated with LPS (10 μg/ml) or poly(I:C) (20 μg/ml) for the indicated time. Whole cell lysates were subjected for immunoblot analysis of phosphorylated (p-) or total level of MAP3K7. Numbers below lanes indicate densitometry of phospho-MAP3K7 relative to total MAP3K7 expression in that same lane. Data are representative of 3 independent experiments with similar results.