Overexpression of Rab7b inhibits LPS-induced production of proinflammatory mediators and LPS-initiated signaling pathways in macrophages. (A) Stable overexpression of His-tagged Rab7b and Rab7b mutants in Raw 264.7 cells. Raw 264.7 cells were transfected with plasmids encoding His-tagged Rab7b, Rab7bT22N, and Rab7b▵CC, and selected under 600 μg/mL neomycin. The stable transfectants were confirmed by RT-PCR (top 2 panels) and Western blotting (bottom 2 panels) using anti-His Ab. (B) Enzyme-linked immunoassay assay of IL-6 and (C) IFN-β production by the indicated Rab7b transfectants after LPS treatments. Cells (2 × 10⁵/well) were treated with 100 ng/mL LPS for 8 hours. The supernatants were then collected and the concentration of (B) IL-6 and (C) IFN-β evaluated by enzyme-linked immunoassay. (D) Western blotting assay of MAPK (E,F), NF-κB, and (F,G) IRF-3 activation after LPS treatments. Raw 264.7 stable transfectants were treated with 100 ng/mL LPS as described for Figure 1B-D. Cell lysates and nuclear proteins were then prepared and blotted with indicated Abs. Total ERK1/2 and β-actin contained in lysates as well as Lamin B contained in nuclear fractions were probed as quantitative controls. (H) NF-κB and (I) 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 Rab7b (mutants)-overexpressing vectors. Luciferase activity was then determined and presented as that described for Figure 2E,F. In panels B, C, H, and I, data are shown as mean (± SE) of triplicate samples. Similar results were obtained in 3 independent experiments. *P < .05; **P < .01.