Figure 2. Unc93b1^S282A attenuates ligand binding and increases association with TLR9.

(a) Biotin-CpG-bound TLR9 complexes were precipitated from lysates of RAW macrophage lines expressing TLR9-HA and the indicated Unc93b1 alleles after stimulation with Biotin-CpG (1µM) for 4h, followed by immunoblot for TLR9-HA. Bars show mean ± s.d. of CpG-bound TLR9 relative to total TLR9; n=3 (WT), n=4 (SKN or S282A), and n=2 (HR), each dot represents an independent experiment. (b) Immunoprecipitation of Unc93b1-Flag from the RAW macrophage lines in (a) followed by immunoblot of TLR9-HA. TLR9 levels in whole cell lysates (WCL) are also shown. Representative of six independent experiments. Bars show mean ± s.d. of TLR9 bound to Unc93b1; n=6 (WT) n=7 (SKN or S282A); each dot represents an independent experiment. (c) Proximity ligation assay to analyze interaction between TLR9-HA and the indicated Unc93b1 alleles in RAW macrophages. Representative images are shown. Control staining was performed by omitting primary anti-HA antibodies. Quantification of PLA signals per cell volume is shown below images. Each dot represents an individual cell. Data are pooled from two independent experiments. Scale bar = 10 µm (d) Immunoprecipitation of TLR9-HA from phagosome preparations of RAW macrophage lines in (a) followed by immunoblot for Unc93b1-Flag. TLR9 and Unc93b1 levels in phagosomes and whole cell lysates are also shown. Representative of two independent experiments. All p-values determined by unpaired two-tailed Student’s t-test.