Extended Data Fig. 10: RIPR-SIRPα development and in vitro testing.

a, b, HEK293 cells were transiently transfected with target receptors and LCK (a), or LCK plus CD45 (b). After lysis, chimeric receptors were immunoprecipitated with anti-HA antibody directly conjugated to magnetic beads. Samples were probed for phosphotyrosine and HA by western blot. Data are representative of three independent biological repeats. For raw source image, see Supplementary Fig. 1. c, RIPR-SIRPα(3C) was treated with 3C (100 μg ml⁻¹) for 14 h at 4 °C. 3C-digestion was analysed by Coomassie blue staining on SDS–PAGE gel. d, Human PBMC macrophages were pretreated with RIPR-SIRPα or ‘Velcro’ protein for 30 min at 37 °C and incubated with human tumour cells (Raji) pretreated with varying concentrations of rituximab (from 0 to 5 μg ml⁻¹) for 30 min at 37 °C. Macrophages were co-cultured with 1 × 10⁴ CFSE+ Raji cells for 2 h at 37 °C. e, Quantification of phagocytosis as described in d for a fixed concentration of rituximab (1 μg ml⁻¹) for cells treated with 100 nM or 500 nM of intact or 3C-digested RIPR-SIRPα or Velcro, as indicated. In d, e, data are mean ± s.d. from n = 2 (d) or n = 3 (e) biological replicates representative from 2 independent experiments. For representative gating strategy, see Supplementary Fig. 2. f, Coomassie blue staining on SDS–PAGE gel for AB21 Fab and RIPR-SIRPα(AB21). g, HEK293 cells were transiently transfected with LCK, CD45, CD45_dead and SIRPα as indicated. HEK293 cells were treated with RIPR-SIPRα 24 h after transfection for 30 min at 37 °C. Cells were collected and cell lysates were incubated with HA beads, HA–SIRPα was immunoprecipitated and analysed by western blot assay as indicated. Data are representative from 2 independent experiments. For raw source image, see Supplementary Fig. 1.

Source data