Modulation of MAPK, NF-κB, mTOR, and Akt signaling in DCs generated in RAPA. Murine CTR- and RAPA-DCs were generated, positively selected using immunomagnetic beads, and cytoplasmic and nuclear proteins isolated after 20 minutes of exposure to 0.1 μg/mL LPS, 1 μg/mL CpG, 5 μg/mL agonistic CD40 mAb, or 0.1 μg/mL IL-33 for for 20 minutes. (A) Western blot analysis revealed that generation of DCs in RAPA limited activation of p38 and JNK after ligation of TLR4, TLR9, and CD40 (IL-33 receptor), but not ST2L. (B) Nuclear extracts were assessed for DNA-binding activity in electrophoretic mobility shift assay using ³²P-labeled NF-κB probes. The data show inhibited NF-κB activation downstream of TLR4 in RAPA-DCs. Vertical lines indicate repositioned gel lanes within the same gel and experiment. (C-D) Generation of DCs in RAPA did not modulate expression of the mTOR kinase or the mTORC1 component Raptor. In addition, generation of mDCs in RAPA blocked mTOR autophosphorylation on S2481 and p70S6K phosphorylation on T389. Vertical lines indicate repositioned gel lanes within the same gel and experiment. (E) Phosphorylation of Akt on T308 was increased in RAPA-DCs and the mTORC2-mediated phosphorylation of Akt on S473 was maintained. Data are representative of at least 2 independent experiments.