Fig. 8
From: A non-conserved amino acid variant regulates differential signalling between human and mouse CD28
Nck SH2 domain is required for Vav1 recruitment to CD28 and actin rearrangement and polarisation. a hCD28WT cells were transfected with pm-Cherry Vav1 (magenta) vector together with GFP-Nck WT (green) or a GFP-Nck mutants within the SH2 domain (R311Q) or within the SH3.2 (W149K) domains and then stimulated for 15 min in the absence (Ctr) or presence of Dap3/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. The frequency of conjugates (total 15) showing significant (RRI > 2.5) Vav1 and Nck recruitment (b, c) or F-actin polarisation (c) were calculated and data represent the mean ± SD of three independent experiments. Mean values: Nck WT, Nck = 5.1 ± 2.6, Vav = 5 ± 2.8, F-actin = 7.2 ± 2.9; Nck R311Q, Nck = 1.1 ± 0.3, Vav = 1.1 ± 0.7, F-actin = 2.8 ± 1.8; Nck W149K, Nck = 4.3 ± 2.8, Vav = 3.9 ± 2.4, F-actin = 5.5 ± 3.9. **p < 0.01, ***p < 0.001 calculated by Student’s t-test compared with Nck WT. The results are representative of three independent experiments
