PIR-B negatively regulates the positive feedback mechanism of IFN-α secretion triggered by TLR9. (A) Flow cytometric analysis of cytoplasmic TLR9 expression on B6 and Pirb^−/− Flt3-L–induced PDCs. Mean fluorescent intensities. (B) B6 and Pirb^−/− PDCs are incubated with CpG-A (OD2216) for 24 hours at the indicated concentration. The amounts of IFN-α are measured by ELISA assay. Data are mean ± SEM (n = 4). The results are representative of 3 separate experiments. Statistical analyses were performed using Student t test: *P < .05. (C-G) Immunoblot analysis of B6 and Pirb^−/− PDCs stimulated with CpG-A (3 μg/mL). (C) Immunoblots of PIR-B phosphotyrosine (4G10), SHP-1, and PIR-B after the precipitation with anti–PIR-A/B. (D) Immunoblots of pErk1/2, Erk, pp38, and p38. (E) Immunoblots of pIκB kinase α- and β-actin. (F) Immunoblots of pBtk and Btk. (G) Immunoblots of pSTAT1, STAT1, pSTAT2, and STAT2. (H-I) Immunoblot analysis of B6 and Pirb^−/− PDCs stimulated with IFN-α (100 U/mL). (H) Immunoblots of PIR-B phosphotyrosine (4G10), SHP-1, and PIR-B after the precipitation with anti–PIR-A/B. (I) Immunoblots of pSTAT1, STAT1, pSTAT2, and STAT2. The immunoblot results are representative of 3 separate experiments.