Binding of aPLAs to TLR2 on HUVECs, HEK293 cells, and monocytes. Binding of aPLAs to TLR2 or TLR4 on monocytes (A), HEK-TLR2 cells and HEK-TLR4 cells (B), and HUVECs and TNF-pretreated HUVECs (TNF; C) was quantified using the proximity ligation assay. Cells were incubated for 1 hour with 100 μg/mL of aPLAs or control IgGs (CTL), and then for 1 hour with 10 μg/mL of polyclonal rabbit anti-TLR2 or anti-TLR4 antibodies. Cells were then incubated with probe-labeled secondary antibodies, and observed for the development of a fluorescent signal, as described in “Quantification of a PLA binding to TLR2 and TLR4 on HEK-TLR2 or HEK-TLR4 on HUVECs and on monocytes.” Binding was evaluated by quantifying the fluorescence area per cell and is expressed in arbitrary units proportional to the experimental condition that gave the highest fluorescence signal for each cell type. The average number of counted cells per image was 60 ± 2 for HEK-TLR2, 59 ± 3 for HEK-TLR4, 40 ± 10 for HUVECs, 42 ± 7 for TNF-treated HUVECs, and 21 ± 6 for monocytes.