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. 2012 Apr 9;287(22):18806–18819. doi: 10.1074/jbc.M112.340935

FIGURE 6.

FIGURE 6.

Eps8 promotes TLR4-MyD88 interaction in macrophages. A, RAW264.7 cells were plated on a glass coverslip overnight, incubated without (top) or with LPS-coated latex beads (bottom) for 10 min, fixed, permeabilized, and stained for Eps8 and TLR4. The confocal images of Eps8, TLR4, and the merged file are shown as indicated. The latex bead, marked by an arrowhead, was observed in bright field. The arrow indicates the phagosome. Scale bar, 4 μm. RAW264.7 (B) and its derived Ctrl and siEps8-346-1 (siEps8-346) cells (C) or MyD88-attenuated (siMyD88) cells (D) were treated with or without LPS (100 ng/ml) for 24 h. The lysates (Input) and the TLR4, Eps8 (Eps8 or N-Eps8), and control (IgG) immunocomplex from each sample were resolved by SDS-PAGE and Western immunoblotting with antibodies as indicated. E, BMDMs from wild type (Wt) and MyD88−/− mice were stimulated without or with LPS for 24 h. Equal amounts of lysates (30 μg) were resolved by SDS-PAGE and probed with antibodies as indicated. The arrow indicates the position of active Src. F, RAW264.7-derived Ctrl and 261-p97Eps8-expressing cells (261-9) were stimulated without or with LPS for 24 h. The lysates (right) and the immunoprecipitates of TLR4 (left) and Eps8 (N-Eps8; middle) from each sample were resolved by SDS-PAGE and Western immunoblotting with antibodies as indicated. Asterisks indicate the position of ectopically expressed 261-p97Eps8. It should be noted that the level of the upper band varied in different experiments. IP, immunoprecipitation.