Figure 7.

Activation of CD11b by LA1 could endocytose TLR4 in macrophages. (A, B) BMDMs were treated with LA1 (20 μM) for 1 and 2 h. FACS analysis was performed to assess the expressions of TLR4 (A) and CD11b (B) in BMDMs. (C, D) C57BL/6 mice were stimulated with LA1 (40 μg/g of body weight) for 1 and 2 h. FACS analysis was performed to assess the expressions of TLR4 (C) and CD11b (D) in PBMCs. (E) Slides containing BMDMs were treated with LA1 (20 μM) for 1 and 2 h, and then, the cells were fixed and permeabilized. The cells were initially stained with anti-TLR4 mAb and anti-CD11b mAb, followed by incubation with an Alexa Fluor 488- and 647-conjugated secondary antibodies. Finally, they were analyzed using a confocal microscope. Red represents CD11b; green represents TLR4; light blue represents nuclei with DAPI; and yellow represents merged TLR4 and CD11b. (F) After treated with LA1 (20 μM) for 1 or 2 h, BMDMs were lysed in a modified RIPA buffer. After entrifuged, one aliquot of the supernatant was saved as the input control, the remainder was separately incubated with anti-CD11b antibody, anti-TLR4 antibody and negative control IgG antibody, followed by pull-down with 30 μl Protein A Agarose beads. The beads were then collected by centrifugation and washed three times with cold PBS. The immunoprecipitates were eluted by boiling in loading buffer and subjected to western blot analysis along with input sample as described above. Data shown are representative of three independent experiments. Error bars represent S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by one-way analysis of variance (ANOVA) post-hoc Tukey Multiple Comparison Test.