Supplementary Figure 8: Ca²⁺ influx enhances the openness and signaling of CD28.

(a-b) To further demonstrate the effect of Ca²⁺ on CD28_CD-membrane binding, we use different concentrations of α-CD3 to generate different levels of Ca²⁺ influx. We also used BAPTA pre-treatment to chelate the intracellular Ca²⁺ (a). The de-quenching FRET was used to detect the effect of TCR-induced Ca²⁺ influx on the CD28_CD-membrane interaction. Different doses of α-CD3 induced different levels of Ca²⁺ influx (a). Treatment of 10 μM BAPTA led to partial chelation of intracellular Ca²⁺. N = 64, 28, 22, 36 (from left to right) for each condition. Each dot represents the FRET value from one individual cell. Mouse IgG (3 μg/ml) was used as the isotype control antibody for α-CD3.

(c) To exam the effect of Ca²⁺ influx on CD28 signaling in primary T cell, the mouse CTL were generated and stimulated with α-CD3 (0.5 μg/ml) + α-CD28 (2 μg/ml) with indicated times in the Ca²⁺/Mg²⁺-free Ringer’s buffer containing 1 mM Ca²⁺ or not at 37 ^oC. After stimulation, cells were lysed for immunoprecipitation and immunoblotting. The bands were quantitated by ImageJ. The pCD28/CD28, P85/CD28, Lck/CD28 and Grb2/CD28 ratios were obtained and further normalized to the value of no stimulation condition of each strip.

Data are representative of three independent experiments. In (b), for the left sub-panel, One-Way ANOVA was used to analyze difference among four groups (P<0.0001) and unpaired t-test was used to analyze difference between two groups. For the middle and right sub-panels, the R18 level of the α-CD3 (3μg/ml) group and the mTFP level of the isotype control group do not fit the normal distribution. To compare each two groups, Mann Whitney test was used for data sets that do not fit the normal distribution and unpaired t-test was used for the rest. The center value and error bar in b denote mean and s.e.m.. The original gel image of b can be found in Supplementary Data Set 2. Source data for b are available in Source Data 9.

Source data