Figure 5. WAVE2 is released from an autoinhibitory conformation following TCR engagement.

(a(i)) Schematic representation of the tagged WAVE2 protein used for FRET analysis. (a(ii)) Jurkat cells expressing YFP-WAVE2-CFP and untransfected cells (negative control – Neg. Cont.) were analyzed by western blotting with anti-GFP. GAPDH served as a loading control. (a(iii)) Jurkat T cells expressing YFP-WAVE2-CFP were plated over either non-stimulatory coverslips coated with anti-CD43 antibody (upper panel, n = 89 cells), or over stimulatory coverslips coated with anti-CD3 antibody (lower panel, n = 42 cells). Cells were fixed following 3 min of activation and imaged by confocal microscopy. FRET analysis was performed using the donor-sensitized acceptor emission technology (see Methods for details). A graph summarizing the mean FRET efficiency in cells expressing YFP-WAVE2-CFP plated over the indicated coverslips is presented on the right. Graph shows means ± SEM from three independent experiments. Significance was determined by two-tailed Student’s t-test. (b(i)) Scheme of 81-183aa-deleted YFP-WAVE2-CFP. (b(ii)) Jurkat T cells transfected with a plasmid encoding YFP-WAVE2-CFP Δ81-183aa were plated over either non-stimulatory (anti-CD43, upper panel, n = 76 cells) or stimulatory (anti-CD3, lower panel, n = 48 cells) coverslips and fixed after 3 min of activation. A graph showing the mean FRET efficiency in cells expressing YFP-WAVE2-CFP Δ81-183aa plated over the indicated coverslips is presented on the right. Graph shows means ± SEM from three independent experiments. Significance was determined by two-tailed Student’s t-test.