Supplementary Figure 7: Ionomycin-induced Ca2+ influx enhances the openness of CD28.
From: Dynamic regulation of CD28 conformation and signaling by charged lipids and ions
The de-quenching FRET method was used to measure the effect of Ca2+ on CD28CD-membrane binding in live Jurkat T cells. Ionomycin (1 μM) was used to trigger Ca2+ influx in T cells. Ca2+ influx led to the significant decrease of the FRET value of CD28-TFP (a), indicating the dissociation of CD28CD from the membrane. In contrast, the FRET values of the 3aa-TFP and 50aa-TFP control constructs were insensitive to Ca2+ influx (b, c). The FRET efficiency was measured and plotted as mean +/- s.e.m. (n = 12, 15 in (A), n = 17, 16 in (B), n = 22, 19 in (C) (from left to right), each dot represents the FRET value from one individual cell). The surface mTFP level and R18 level were comparable.
Data are representative of two independent experiments. All the data were analyzed by unpaired t-test except the left sub-panels in (a) and (c) where Mann-Whitney test were used because the FERT efficiency data sets of the CD28 group and the 50aa-TFP group do not fit the normal distribution. The center value and error bar denote mean and s.e.m.. Source data are available in Source Data 8.
