TLR4 is internalized and transported to Rab7b-positive compartments after LPS treatment. (A-D) TLR4 translocation after LPS treatments. Raw 264.7 cells were transiently transfected with TLR4-HA vector; 48 hours later, the cells were left untreated or treated with 100 ng/mL LPS for indicated period. The subcellular localization of TLR4 was examined after immunostaining for TLR4 and indicated compartments markers under confocal microscopy. (E) Colocalization of TLR4 with Rab7b after LPS treatment. Raw 264.7 cells were transiently cotransfected with TLR4-HA with His-Rab7b plasmids, and treated with 100 ng/mL LPS for 30 minutes to 1 hour. Then the cells were immunostained with anti-HA Ab and anti-His Ab, manifested with Orange Green 488-conjugated or Alexa Fluo 555-conjugated secondary Ab. Representative images are shown in (A-E). (Bar = 100 μm) (F) Subcellular fractionation of Rab7b-overexpressed Raw 264.7 cells. Raw 264.7 cells stably overexpressing Rab7b were fractionated for early endosomes (EE) and late endosomes/lysosomes (LE/Lyso). Distribution of specific markers as well as Rab7b in each fraction was examined by Western blotting. (G) Immunoisolation of EE and LE/Lyso. Fractions obtained in (F) were pooled for immunoisolation of EE (fractions 1-4) and LE/Lyso (fractions 8-12). The EE was enriched by using anti-TfR/CD71 Ab while LE/Lyso was enriched by using anti-LAMP-1 Ab. Rabbit IgG was used as negative control. The immunoisolated organelles were examined for indicated markers, showing the specificity of immuno-isolated EE and LE/Lyso. (H) Dynamic translocation of TLR4 from EE to LE/Lyso after LPS treatments. The indicated transfectants were treated with 100 ng/mL LPS for indicated time periods, and the EE and LE/Lyso organelles were immunoisolated and subjected to Western blot assay of TLR4 expression. EEA-1 and LAMP-1 were examined to determine the loading quantity of the immunoisolated organelles. Similar results were obtained in 3 independent experiments.