Inefficient uptake of CpG and retarded redistribution of TLR9 in Ly49Q^lo cells. (A) Expression of Ly49Q in RAW264 clones. Ly49Q^hi or Ly49Q^lo RAW264 clones were established from bulk RAW264 cells by limiting dilution. Four clones of each type were analyzed, and similar results were obtained. Data from representative clones are shown. (B) No difference in TLR9 and TLR4 expression in RAW264 cells. Semiquantitative RT-PCR was carried out using RNA prepared from the indicated cells. The presence or absence of Ly49Q had no effect on the transcription of these TLRs. In the photographs, the PCR templates were sequentially diluted by 5-fold. (C) Quantitative analysis of IFN-β transcription in RAW264 cells in response to CpG. Ly49Q^hi or Ly49Q^lo RAW264 cells were stimulated with CpG1668 (0.3 μM) for 4 hours, and quantitative RT-PCR analyses were performed. The histograms show the relative amounts of IFN-β mRNA evaluated by real-time PCR. (D) Intracellular redistribution of CpG and TLR9. TLR9-GFP expression plasmids were introduced into RAW264 cells. Twenty-four hours after transfection, the cells were incubated with rhodamine-conjugated CpG (0.3 μM) for the indicated periods. Impaired CpG/TLR9 redistribution was observed in the Ly49Q^lo RAW264 cells. (E) Internalization and distribution of CpG in RAW264 cells. RAW264 cells were incubated with rhodamine-conjugated CpG (0.3 μM) and fixed at the indicated time points. (F) Intracellular distribution of rhodamine-conjugated CpG. After 24 hours of incubation with CpG, tubular endolysosomal structures were observed in Ly49Q^hi RAW264, but not in Ly49Q^lo RAW264 cells.