Activation of signaling pathways in B cells and BMDC from WT and ABIN1[D485N] mice. (A–C) B cells from WT and ABIN1[D485N] mice were stimulated with 0.5 µg/ml of the TLR7 agonist R848 (A), 10 µg/ml α-IgM (B), or 10 µg/ml α-CD40 (C) for the times indicated, and cell lysates were probed with the antibodies indicated. *, nonspecific band. Antibodies that recognize GAPDH and p38 MAPK served as loading controls. The α-phospho (p)–IKK-α/β antibody in C–E (Ser176/180, antibody 16A6; Cell Signaling Technologies) differed from that used in A and B (Ser180/181; Cell Signaling Technologies). (D and E) BMDC from WT and ABIN1[D485N] mice were stimulated with 0.25 µg/ml of the TLR7 agonist R848 (D) or 2 µg/ml of the TLR2/6 agonist LTA (E) for the times indicated, and cell lysates were probed with the antibodies indicated. (A–E) The data are representative of two to four independent experiments. (F) BMDC were stimulated with 0.5 µg/ml R848 for the times indicated and lysed, and TAK1 was immunoprecipitated with α-TAK1 and assayed as described in Materials and methods. The activity is plotted as fold increase compared with that measured in the lysates from cells not been stimulated with R848. The results are average data from three different experiments, each with BMDC from different mice, with each sample being assayed in duplicate. Error bars represent mean ± SEM.