Figure 5.

Human PLT TLR9 localization with VAMPs 5, 7, and 8 under resting conditions and when activated (spread) on glass surface. Samples were examined by confocal fluorescence microscopy. Image analysis was completed by using the JACoP (Just Another Colocalization Plugin) plugin for ImageJ as described in Table 1. Manders’ coefficients were used to compare the TLR9 relationship to the associated VAMP. (A) Micrographs demonstrating the relationship of VAMP 5 and TLR9. As demonstrated in the micrographs, in the resting PLT, ∼41% of TLR9 signal overlaps with that of VAMP 5. The portion of overlapping signal slightly increased in the adhered PLTs to 49%. (B) Additional micrographs demonstrating the relationship between VAMP 7 and TLR9. Image analysis confirms 82% of TLR9 overlapped with that of VAMP 7 in the resting PLT; however, upon spreading, the signal decreased to only 56%. (C) Micrographs of resting and spread PLTs probed with specific antiserum for VAMP 8 and TLR9. In the resting PLT, 77% of TLR9 signal overlapped with VAMP 8. Upon spreading, the signal decreased to 67% signal overlap.