IRAK-2 is required for early TLR2 responses in MOLF/Ei but not C57BL/6J macrophages. (A–C) C57BL/6J and MOLF/Ei BMDMs were infected with lentiviral constructs containing either control or one of three IRAK-2–targeting shRNA sequences. (A) To assess knockdown efficiency, mRNA was harvested and cDNA was amplified with IRAK-2 or GAPDH-specific PCR primers. (B and C) Cells were stimulated with 2 µg/ml LTA and IL-6 was quantified at 4 h using RT-PCR, with error bars indicating SEM (B), or at 6 h using ELISA, with error bars indicating SEM (C). n.d., not detected. Data are representative of three independent experiments. (D and E) MOLF/Ei BMDMs were infected with control or IRAK-2–targeting construct number two and stimulated with 2 µg/ml LTA (D) or under hypertonic NaCl conditions (E) for the indicated times. MAPK and NF-κB pathway signaling events were assessed using the indicated Western blot analyses. Total p38 and ERK serve as loading controls. Data are representative of at least two independent experiments.