Journal: bioRxiv

Article Title: An essential checkpoint for TLR9 signaling is release from Unc93b1 in endosomes

doi: 10.1101/410092

Figure Lengend Snippet: ( a ) Unc93b1 SKN and Unc93b1 S282A attenuate ligand binding of TLR9. Streptavidin immunoprecipitation of lysates from RAW macrophage lines, expressing TLR9-HA and the indicated Unc93b1 alleles, stimulated for 4h with biotinylated CpG-B (1μM) followed by immunoblot for TLR9. Bar graph shows quantification of CpG-bound TLR9 over total TLR9 from several experiments. ( b ) Unc93b1 SKN cells show normal DNA uptake. Uptake of Cy3-labeled CpG-B (1μM) as measured by flow cytometry of RAW macrophage lines expressing the indicated alleles of Unc93b1. ( c ) Unc93b1 SKN and Unc93b1 S282A bind stronger to TLR9. Flag immunoprecipitation of Unc93b1 from RAW macrophage lines, expressing TLR9-HA and the indicated Unc93b1 alleles, followed by immunoblot of TLR9. Input levels of TLR9 in whole cell lysates (WCL) are also shown. Bar graph shows quantification of TLR9 bound to Unc93b1 from several experiments. ( d ) HA immunoprecipitation of TLR9 from phagosome preparations of RAW macrophage lines, expressing TLR9-HA and the indicated Unc93b1 alleles, followed by immunoblot for Unc93b1. Input levels of TLR9 and Unc93b1 in phagosomes and whole cell lysates (WCL) are also shown; FL: full-length. Representative of two independent experiments. All data are mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t-test. The data are representative of at least three independent experiments, unless otherwise noted.

Article Snippet: Streptavidin Magnetic Beads for biotin pull downs and Protein G agarose were purchased from Pierce.

Techniques: Ligand Binding Assay, Immunoprecipitation, Expressing, Western Blot, Labeling, Flow Cytometry