Fig. 6
From: A non-conserved amino acid variant regulates differential signalling between human and mouse CD28
P212 within the C-terminal proline-rich motif of human CD28 is essential for Vav1 recruitment and actin polymerisation. a hCD28 WT or hCD28P212A or chimera containing the extracellular and transmembrane domain of human CD28 and the cytoplasmic tail of mouse CD28 (Chimera CD28WT) or mouse CD28 mutant (Chimera CD28A210P) were transfected with 20 μg of GFP-Vav1 (green) and then stimulated for 15 min in the absence (Ctr) or presence of Dap/B7 cells (B7). After fixing and permeabilisation F-actin was stained with 633-conjugated phalloidin (blue) and analysed by confocal microscopy. The scale bar represents 10 μm. b, c The relative recruitment index (RRI) of Vav1 (b) and F-actin (c) was calculated as described in Methods and represents the mean ± SD of 15 conjugates analysed in each group. Mean values: Vav, hCD28WT = 3.6 ± 1.6, hCD28P212A = 1.3 ± 0.24, Chimera CD28WT = 1.14 ± 0.2, Chimera CD28A210P = 2.33 ± 0.9; F-actin, hCD28WT = 8.65 ± 3.6, hCD28P212A = 2.2 ± 0.9, Chimera CD28WT = 2.9 ± 1, Chimera CD28A210P = 7.6 ± 3.8. Asterisks **p < 0.01, ***p < 0.001 calculated by Student’s t-test compared with hCD28WT. The results are representative of three independent experiments
