Supplementary Figure 6: Disruption of CD28CD-membrane binding promoted the protein’s basal signaling.
From: Dynamic regulation of CD28 conformation and signaling by charged lipids and ions
(a) The de-quenching FRET was used to measure the membrane binding of CD28 WT and CD28 linker mutant (n = 21, 38 (from left to right), each dot represents the FRET value from one individual cell).
(b-e) TFP-T2A-HA-CD28 WT or linker mutant construct was transduced into mouse CD28−/− T cells by retrovirus, respectively. After protein translation of the fusion construct, TFP and HA-CD28 were separated due to the self-cleaving property of T2A. Basal CD28 signaling was measured in transduced cells without stimulation. IL-2 production was measured in transduced cells under TCR and/or CD28 stimulation.
(b-c) The cells without stimulation were lysed and the immunoprecipitation assay was performed to detect basal phosphorylation of CD28 and its interactions with signaling proteins. The bands were quantitated by ImageJ. The pCD28/HA, P85/HA, Lck/HA and Grb2/HA ratios were obtained and further normalized to the value of WT condition of each strip. Average results of 4 independent samples are shown in panel C.
(d-e) The cells were stimulated for 4 hours with plated-bound α-CD3 (0.5 μg/ml) alone or α-CD3 (0.5 μg/ml) + α-CD28 (2 μg/ml), and IL-2 production was measured by intracellular staining and flow cytometric analysis. CD4+ TFP+ cells were gated for the analysis of IL-2 level. TFP levels of CD28 WT cells and CD28 linker cells were matched, reflecting the comparable expression level of CD28 WT and CD28 linker. Percentage of IL-2 positive cells and median fluorescence intensity (MFI) of all cells are shown (n = 3).
Data are representative of three (a, c) or four (e) independent experiments, and were analyzed by unpaired t-test. The center value and error bar in a, c and e denote mean and s.e.m.. The original gel image of b can be found in Supplementary Data Set 2. Source data for a, c and e are available in Source Data 7.
