Figure 3. Ligand binding assay reveals SR-A ligand(s) in macrophage:tumour cell co-culture supernatants.

A. An ELISA based ligand binding assay was used to screen for a possible SR-A ligand in co-culture supernatants. Supernatant from ID8, PANC02, wt or SR-A^-/- macrophages or embryonic mouse fibroblasts (EMF) alone showed no ligand binding. Co-culture supernatant from wt or SR-A^-/- macrophages with ID8 or Panc02 cells showed a significant increase in SR-A binding activity (p<0.01, t-test). There was no SR-A ligand binding detectable in the supernatant from macrophages with EMF. AcLDL was used as a positive control. Data are represented as mean ± SD of n=12. Representative data are shown from at least 3 independent experiments. B. SR-A binding activity is lost after repeated passaging of co-culture supernatants on wt but not SR-A^-/- BMM monolayers. Co-culture supernatants were incubated twice or five times for 1h with wt or SR-A^-/- BMMs, then SR-A ligand activity in the passaged supernatant was assessed by ELISA as in A. Data are represented as in A. C. Co-culture supernatants contain a lipid ligand. Supernatants as in B. were treated with PMH-Liposorb resin to remove all lipids or lipid-bound molecules, and then tested for SR-A binding activity by ELISA as in A.