Figure 2.

Collaboration of Syk kinase and TLR/MyD88 pathways sustain IkB degradation and enhance NFkB nuclear translocation. TNF production from C57BL/6 wild-type and MyD88^–/– (A) or BALB/C wild-type and Syk^–/– (B) macrophages stimulated with 10 μg/mL particulate β-glucan and 10 ng/mL Pam₃CSK₄, as indicated. (C) RAW macrophages stimulated with 10 ng/mL Pam₃CSK₄ and 10 μg/mL particulate β-glucan. Top: IkB degradation after 2 h was assayed by Western blotting. Bottom: localization of NFkB c-Rel in nuclear lysates. Numbers above each lane show fold decrease (IkB) or increase (cRel) of the relative band intensities of IkB and c-Rel, with actin and USF-2 as loading controls, versus unstimulated control. (D) IkB degradation in RAW macrophages was assayed after the indicated times. Data shown are mean ± SD and are representative of two independent experiments. (E) Nuclear translocation of c-Rel (green) following costimulation of C57BL/6 macrophages with 10 ng/mL Pam₃CSK₄ and 10 μg/mL particulate β-glucan for 1 h. Nuclei were stained with Hoechst 33258 (red). Scale bar represents 10 μm. (F) Nuclear translocation of c-Rel was quantified microscopically over time in C57BL/6 macrophages stimulated with 10 ng/mL Pam₃CSK₄ followed by 10 μg/mL particulate β-glucan. Data shown are mean ± SD and are representative of two independent experiments.