Rab7b negatively regulates the TLR4 expression. (A,B) Western blotting assay of TLR4 expression in Rab7b-overexpressed or (C,D) Rab7b-silenced peritoneal macrophages (A,C) and Raw 264.7 cells (B,D). Stable Raw 264.7 transfectants (B,D) and murine peritoneal macrophages (A,C) transiently transfected with indicated vectors were prepared for cell lysates and TLR4 expression examined by Western blotting using anti-TLR4 Ab. β-actin was used as quantitative control. The expression level of Rab7b by these transfectants was determined by quantitative PCR, and the mean folds of Rab7b mRNA level to that of β-actin were shown at the top of each panel. (E,F) FACS assay of TLR4 expression on plasma membrane of Rab7b-silenced (lower panels) or control vector-silenced (upper panels) murine peritoneal macrophages and Raw 264.7 cells. TLR4 expression on plasma membrane was evaluated by labeling cells with phycoerythrin-conjugated TLR4 Ab. The mean fluorescence intensity (MFI) was also shown at the upper right corner of each panel. (G) Dynamic changes of TLR4 expression in Rab7b-silenced peritoneal macrophages and Raw 264.7 cells after LPS treatments. Cells were transiently transfected with control silencing small RNAs (Ctrl siRNA1) or Rab7b silencing small RNAs (Rab7b siRNA1); 48 hours later, cells were treated with 100 ng/mL LPS. TLR4 contained in cell lysates were examined by Western blotting using TLR4 Ab. β-actin was used as quantitative control. Similar results were obtained in 3 independent experiments.