Silencing of Rab7b expression promotes LPS-initiated signaling pathways in macrophages. (A) Stable silencing of Rab7b expression in Raw 264.7 cells. Raw 264.7 cells were transfected with plasmids encoding small RNAs (Ctrl siRNA1 and Rab7b siRNA2), and selected under 600 μg/mL neomycin. The efficiency of silencing was evaluated by quantitative PCR as described in Figure 1A-C. (B) Western blotting assay of MAPK and (C) NF-κB activation after LPS treatments. Control (Ctrl siRNA1)-stably silenced or Rab7b (Rab7b siRNA1)-stably silenced Raw 264.7 cells were treated with 100 ng/mL LPS as indicated. Then cell lysates were prepared and blotted with indicated Abs. (D) Western blotting assay of IRF-3 activation after LPS treatments. Cells in panels B and C were extracted for lysates and nuclear proteins. Then p-IRF-3 contained in lysate (lysate) and total IRF-3 contained in the nuclei (nuclear) were examined by Western blotting. In panels B-D, total ERK1/2, p38, and β-actin contained in lysates as well as Lamin B contained in nuclear fractions were probed as quantitative controls. (E) NF-κB and (F) IFN-β luciferase reporter assay. RAW264.7 macrophages in 24-well plates were transiently cotransfected with 10 ng of pTK–Renilla-luciferase, and indicated amounts of GL3.5XκB-luciferase or IFN-β luciferase reporter plasmids and silencing vectors (pCtrl siRNA1 or pRab7b siRNA1). Total amounts of plasmid DNA were equalized using corresponding control vector (mock). After 24 hours of culture, the cells were stimulated with 100 ng/mL LPS for 8 hours. NF-κB luciferase activity (E) and IFN-β luciferase activity (F) were measured using the Dual-Luciferase Reporter Assay System, normalized by Renilla luciferase activity, expressed as fold induction relative to the activity in unstimulated cells transfected with control vector. In panels E and F, data are shown as mean (± SE) of triplicate samples. Similar results were obtained in 3 independent experiments. ▴P < .05; *P < .05; **P < .01; ***P < .001.