Supplementary Figure 9: CD28 ligation caused the dissociation of CD28 cytoplasmic domain from the membrane but was insufficient to induce CD28 phosphorylation.
From: Dynamic regulation of CD28 conformation and signaling by charged lipids and ions
(a) The de-quenching FRET method was used to measure whether CD28 ligation could induce CD28 cytoplasmic domain to dissociate from the membrane in live Jurkat T cells. The stimulating buffer was Ca2+/Mg2+-free Ringer’s buffer. Compared with mock treatment (no antibody), addition of 5 μg/ml α-CD28 induced dissociation of CD28CD from the membrane. To test whether this induction was dependent on the phosphorylation of CD28, 100 μM PP2 was pre-incubated with T cells for 10 min to inhibit Src family kinase and then the cells were stimulated by 5 μg/ml a-CD28. The result indicated CD28 phosphorylation was not required for the dissociation of CD28CD from the membrane. N = 22, 11, 12 for each condition (from left to right). Each dot represents the FRET value from one individual cell. Data are representative of three independent experiments. Unpaired t-test was used to analyze difference between two groups.
(b) CD28-deficient Jurkat T cells expressing HA-mCD28 were stimulated by 1 μg/ml a-CD3 alone, 2 μg/ml a-CD28 alone or 2 μg/ml a-CD28 and 1 μg/ml a-CD3 in normal Ringer’s buffer (containing 1 mM Ca2+) for the indicated time at 37 oC. Either CD3 ligation alone or CD28 ligation alone could not trigger CD28 phosphorylation, but CD28 ligation and TCR ligation together could trigger CD28 phosphorylation. The bands were quantitated by Image J. The pCD28 band intensity was divided by the corresponding CD28 band intensity to obtain the pCD28/CD28 ratio, which were further normalized to the value of 0 min of α-CD3 condition.
The center value and error bar in a denote mean and s.e.m.. The original gel image of b can be found in Supplementary Data Set 2. Source data for a are available in Source Data 10.
