Fig. 2 | Nature Communications

Fig. 2

From: Engineered proteins with sensing and activating modules for automated reprogramming of cellular functions

Fig. 2

The activation and phagocytic functions of SIRPα Shp2-iSNAP in macrophages. a Schematic representation of the drawing of the SIRPα Shp2-iSNAP and its putative activation mechanism upon CD47 engagement. SIRPα Shp2-iSNAP contains a human SIRPα without its ITIM-containing C-tail, fused to the Shp2-iSNAP. b Images of RAW264.7 macrophages expressing SIRPα Shp2-iSNAP or FF mutant after incubation with beads coated by CD47 plus IgG, CD47 only, or IgG only, respectively. c Quantification of local FRET response in macrophages as represented in b (n = 32, 16, 13, 12). d The FRET ratio of SIRPα Shp2-iSNAP in response to CD47-coated beads and 10 μM SFKs inhibitor PP1 treatment in macrophages (n = 22, 22, 22). The same colored dots represent data obtained from the same cell. e The time course of FRET and ECFP fluorescence intensities of one cell from d. The increase of FRET intensity is greater than that of ECFP, so the calculated FRET/ECFP ratio increases compared to before CD47 beads addition. f Images of phagocytosis of opsonized RBCs by a representative RAW264.7 macrophage expressing SIRPα Shp2-iSNAP at indicated time points. Arrows indicate ingested RBCs. g Dot plot of normalized phagocytic rate (mean ± s.e.m.) of macrophages expressing SIRPα Shp2-iSNAP or its controls against opsonized RBCs (‘FF’, phenylalanine mutations replacing two tyrosines in the BTAM peptide; ‘ΔPTP’, SIRPα Shp2-iSNAP without PTP domain; ‘SIRPα’, full-length SIRPα fused with YPet; ‘SIRPα-no ITIM’, ITIM-truncated SIRPα fused with YPet) (n = 7, 7, 4, 7, 6, 6). *P < 0.05, ***P < 0.001 (two-sided Mann–Whitney U-test adjusted for multiple group comparison). Scale bars, 20 μm

Back to article page