Extended Data Fig. 3: The Rh5–CyRPA–Ripr complex does not stimulate Ca²⁺ flux across the erythrocyte membrane.

a, FACS kinetic plot of online stimulation of Fluo-4-loaded erythrocytes, stimulated with a dilution series of the Ca²⁺ ionophore A23187 (1 μM–0.031 μM). An equivolume of a twofold concentration of A23187 was added to erythrocytes loaded with Fluo-4 at 10 s, and fluorescence was monitored continuously for 1 min 30 s. An equivolume of a 2× concentration of Rh5 only or preassembled Rh5–CyRPA–Ripr complex was also added at 10 s after the start of acquisition. Rh5- and Rh5–CyRPA–Ripr complex-bound erythrocytes were then re-measured at 4 min and 6 min. At 7 min post-addition of Rh5–CyRPA–Ripr, Fluo-4-loaded erythrocytes were re-challenged with 1 μM of A23187. b, Kinetic plot of samples to which Rh5 alone or Rh5–CyRPA–Ripr complex was added, plotted only with stimulation with 0.031 μM A23187. c, Representation of mean fluorescence intensity values at 80 s, as above. Experiments were repeated three times with biologically independent samples, and were reproducible.