Figure 5

Single-molecule co-localization between EGFP tagged caspase-4 (C258A) (or Δ80 caspase-4 (C258A)) and AX568-LPS. Caspase-4 (C258A)-EGFP (or Δ80 caspase-4 (C258A)) and AX568-LPS were imaged after immobilization of 100 pM of Caspase-4 with anti-GFP followed by incubation with 6 µM LPS in the presence of 1 µM of AX568-LPS. (a) Schematic of the single-molecule co-localization assay and surface modification. (b) Upper panel: Representative images (25 × 25 µm) for caspase-4 (C258A)-EGFP (left) and AX568-LPS (middle), and their overlapped images (right). Lower panel: Representative images (25 × 25 µm) for Δ80 caspase-4 (C258A)-EGFP (left) and AX568-LPS (middle), and their overlapped images (right). Scale bar, 5 µm. (c) Average number of fluorescent spots for caspase-4 (green), LPS (red), and co-localized caspase-4 (C258A) (or Δ80 caspase-4 (C258A)) and LPS (orange) per image. ± indicates standard error of mean (SEM). (d) Average co-localization efficiency was determined by calculating the number of co-localized spots divided by the number of caspase-4 (C258A) (or Δ80 caspase-4 (C258A)) spots.