Fig 2: Proximity ligation assay (PLA) was employed to confirm proximity of enriched proteins (A-C) integrin α1, (D and E) HLA-E and (F-H) plexin B2. (A) SK-BR-3 cells were pre-incubated with anti-B7-H4 to induce clustering showing proximal integrin α1; (B) prefixed cells showing no surface integrin α1. (C) No B7-H4 antibody negative control (background). (D) Cells pre-incubated with anti-B7-H4 showing co-localised HLA-E; (E) cells not pre-incubated with anti-B7-H4 showing disperse HLA-E. (F) Cells pre-incubated with B7-H4 showing plexin B2 not co-localising with B7-H4 on the cell surface. (G) Cells with anti-B7-H4 only (negative control). (H) Plexin B2 (red) co-localising (yellow) with B7-H4 (green) intracellularly following permeabilization. All images are bright field with red overlay, except E. Scale bars: 10 µm, unless otherwise indicated. Images are representative from 3 replicate experiments.

Fig 3: B7-H4 presence (A) in permeabilised SK-BR-3 cells and (B) on the cell surface of SK-BR-3 cells. (C) Flow cytometry analysis of SK-BR-3 cells where blue represents non-specific antibody (isotype control) and pink is anti-B7-H4 to confirm plasma membrane association, (D) the co-localisation of B7-H4 and biotin after SPPLAT, and (E) Coomassie-staining of proteins eluted from neutravidin beads (lanes 1-3) and streptavidin-HRP blot to confirm biotinylation of eluted proteins (lanes 4-6). Lanes 1 and 4, SPPLAT labelling with HRP-tagged non-specific IgG, lanes 2 and 5, SPPLAT labelling with HRP-tagged anti-B7-H1, lanes 3 and 6, SPPLAT labelling with HRP-tagged anti-B7-H4. Images are representative from 3 replicate experiments. SPPLAT, specific proteomic proximity labelling assay using tyramide; HRP, horseradish peroxidase.