Fig. 6.
NF-κB pathway and lipid rafts are involved with enhanced TLR9 signaling in Abca1-M/-M macrophages. A: BMDMs were incubated with 1 μM CpG for 0–3 h. Cell lysates were harvested and the proteins were analyzed by immunoblotting with the indicated antibodies. IκBα was quantified and normalized to β-actin. The degradation of IκBα induced by CpG stimulation is presented as fraction of basal protein level in control WT cells. B: BMDMs were fractioned into raft and nonraft fractions using a simplified nondetergent method. The protein distribution in each fraction was analyzed by Western blotting using the indicated antibodies. C: BMDMs were treated with 1 μM CpG for 1 h and then lipid rafts (fractions 1–8) and nonrafts (fractions 10–13) were pooled, and the distribution of the indicated proteins was analyzed by Western blotting. D: Lipid raft and nonraft fractions isolated as described in panel C were pooled and immunoprecipitated with anti-TLR9 polyclonal antibody, followed by immunoblotting with anti-TLR9 polyclonal antibody. Results are representative of two independent experiments. E: Lipid raft TLR9 content in macrophages was calculated as a percentage of total TLR9 (lipid rafts + nonrafts). Data are presented as mean ± SEM; n = 3. Statistically significant differences between groups are indicated (* P < 0.05). +/+: WT, -M/-M: Abca1-M/-M.