Y870A TLR9 is not retained in the ER or Golgi.

<p><i>Tlr9</i>^<i>-/-</i> bone marrow was transduced with doxycycline inducible WT or Y870A HA-tagged TLR9, and differentiated towards DCs. Three days later, protein expression was induced by doxycycline (0.5μg/ml) treatment, and BMDCs were fixed and permeabilized at 4 hours post-Doxycycline treatment. The 4 h post-doxycycline fixed cells were labeled for HA and either the late endosome/lysosome marker LAMP1 (A), the ER marker Calreticulin (B), or the Golgi marker GM130 (C). In all cases, nuclei were labeled by DAPI. Each panel shows an image of both WT and Y870A-expressing BMDCs for each individual label, a merged image (Merged), and a colocalization image in which areas of overlap of the two markers, after thresholding by the method of Costes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200913#pone.0200913.ref024" target="_blank">24</a>], is indicated by the white areas (“co-loc. pixels”). HA colocalization with each organelle marker is expressed both as a Pearson’s correlation coefficient and by the percent of manually thresholded HA-labeled structures that overlap with the marker. *, p<0.01; **, p<0.005; ***, p<0.0001.