IRAK-2C is differentially expressed between MOLF/Ei and C57BL/6J macrophages and inhibits NF-κB activity. (A) BMDMs from MOLF/Ei and C57BL/6J mice were stimulated for the indicated times with 2 µg/ml LTA and IRAK-2C mRNA was assessed using real-time PCR primers with a forward primer located within the unique 5′ untranslated region of IRAK-2C and a reverse primer located within IRAK-2 exon 6. Primer specificity was confirmed by agarose gel electrophoresis and sequencing (not depicted). (B and C) Peritoneal macrophages from the indicated strains were stimulated for the times shown with 2 µg/ml LTA. IRAK-2C mRNA was quantified as in A and total IRAK-2 mRNA was quantified using primers common to all isoforms (C). Error bars indicate SEM. (D) RAW264.7 cells were transduced with a lentiviral construct coding for the MOLF/Ei allele of IRAK-2C. Cells were stimulated for the indicated times with 2 µg/ml LTA, and NF-κB and MAPK pathways were assessed by Western blot analysis. Total p38 and ERK served as loading controls. All data are representative of two to four independent experiments.