Fig. S7

Intracelullar activation and localization of zebTLR9 and zebTLR21. (A) HEK293 cells were cotransfected with expression vector for zebTLR9 (Left) and zebTLR21 (Right) and NF-κB luciferase reporter gene. These cells were pretreated with 2 μM chloroquine, and then treated with 0.2 μg/mL flagellin and 3 μM CpG-ODN. Relative luciferase activities were determined. Data are mean ± SD (n = 3). *P < 0.05 vs. cells without chloroquine treatment. (B) HEK293 cells were transfected with expression vector for zebTLR9 and zebTLR21. Expression of zebTLR9 and zebTLR21 was visualized by immunofluorescence staining using anti- FLAG M2 mAb, followed by an Alexa Fluor 488-labeled anti-mouse antibody. Cellular organelles were stained by anti-Lamp1, anti-AIF, anti-EEA1, anti-RCAS1, and anti-calnexin antibodies, followed by an Alexa Fluor 594-labeled secondary antibody. Nuclei were stained by DAPI. Representative merged confocal images are shown for the cellular localization of zebTLR9 and zebTLR21. Green indicates zebTLR9 or zebTLR21; red, organelle markers; blue, nuclei stained with DAPI.