STAT1 is phosphorylated independently of type I IFN after TLR7 triggering with either ligand. GEN2.2 cells were untreated or stimulated with Flu, CL097, or IFN-α for 30 minutes, 2 hours, and 3 hours. Whole-cell protein extracts were prepared. (A) Phospho-STAT1 (pY701) was quantified in the protein extracts by CBA. Data shown are representative of 2 independent experiments. (B) Western blot analysis of phospho-STAT1 (pY701) and phospho-STAT2 (pY690) after activation of GEN2.2 cells. Data shown are representative of 2 independent experiments (C) After 2 hours of stimulation, cells were fixed and permeabilized for phospho-STAT1 (pY701) analysis by flow cytometry. Representative dot plots of 4 independent experiments are shown. (D) Activation of GEN2.2 cells in the absence or presence of anti–IFN-α/β and anti–IFN-α/βR neutralizing antibodies for 2 hours. Phospho-STAT1 (pY701) was analyzed by flow cytometry. Data shown are the means and SDs from duplicate values of 2 independent experiments.