Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Enhanced susceptibility to chemically induced colitis caused by excessive endosomal TLR signaling in LRBA-deficient mice
Figure Lengend Snippet: Enhanced PI3K/AKT/mTOR signaling in Lrba −/− BMDCs and BMpDCs. ( A ) Immunoblot analysis of FOXO1 and FOXO3 in Lrba +/+ and Lrba −/− BMpDCs after stimulation with CpG-A. ( B and C ) Immunoblot analysis of phosphorylated and total mTOR, AKT, S6, and 4E-BP1 in Lrba +/+ , Lrba +/− , and Lrba −/− ( B ) BMpDCs and ( C ) BMDCs stimulated with CpG-A for 16 h ( B ) or for the indicated times ( C ). ( D , Left ) Flow cytometry of Lrba +/+ and Lrba −/− splenocytes, assessing expression of p-AKT and p-S6 by CD11c + DCs. ( D , Right ) Quantification of p-AKT and p-S6 mean fluorescence intensity (MFI) on CD11c + DCs. ( E and F ) Concentration of IFN-α in the culture supernatant of ( E ) BMDCs or ( F ) BMpDCs pretreated with PI3K and mTOR inhibitors for 3 h, then stimulated with CpG-A for 16 h ( n = 3 independent cultures of each genotype from separate mice). ( G ) RT-qPCR analysis of the indicated mRNAs in Lrba +/+ and Lrba −/− BMpDCs after stimulation with CpG-A for 16 h. ( H ) Protein array analysis of the culture supernatant from BMpDCs stimulated with CpG-A. Each symbol ( D ) represents an individual mouse. * P
Article Snippet: Peritoneal m acrophages were stimulated for 16 h with the following TLR ligands: poly (I:C) (200 μg/mL; Invivogen), R848 (20 ng/mL; Enzo Life Sciences), LPS (10 ng/mL; Enzo Life Sciences), CpG-A (100 μg/mL; Invivogen), or CpG-B (200 ng/mL; Invivogen).
Techniques: Flow Cytometry, Cytometry, Expressing, Fluorescence, Concentration Assay, Mouse Assay, Quantitative RT-PCR, Protein Array