Fig. 4
From: A non-conserved amino acid variant regulates differential signalling between human and mouse CD28
P212A substitution within the C-terminal proline-rich motif (YQPP212) of CD28 impairs NF-κB activation. a Amino acid sequence of mouse CD28 WT (mCD28WT) and mCD28 A210P mutant. The substitution A210P in mCD28 is indicated in bold. b–d FACS analysis of Dap3 cells transfected with human B7.1 (Dap/B7) (b) or mouse RAW 264.7 cells (Raw) (c) stained with FITC-conjugated isotype control (Iso) or anti-B7.1 Abs, or CH7C17 cells transfected with mouse CD28 (mCD28) WT or mCD28 A210P mutant stained with phycoerythrin (PE)-conjugated isotype control (Iso) or anti-CD28 (CD28-PE) Abs (d). e–k NF-κB luciferase activity of CH7C17 cells expressing hCD28WT (e–h), or hCD28P212A mutant (e, f) or mCD28WT (g–i) or mCD28A210P mutant (i) stimulated with B7.1 expressing cells (e, g) or agonistic human CD28.2 (f) or mouse 37.51 (h, i), or superagonistic human ANC28.1 (f) or mouse D665 (h). The results are expressed as the mean of luciferase units ± SD after normalisation to GFP expression. j Anti-phosphotyrosine (pTyr) and anti-GAPDH western blotting on total lysates of CH7C17 cells expressing mCD28WT or mCD28A210P mutant stimulated for 10 min with agonistic mouse 37.51 Ab. The position of molecular weight markers, expressed in kDa, is indicated on the right. k IL-8 mRNA levels of CH7C17 cells expressing mCD28WT or mCD28A210P mutant stimulated for 6 h with superagonistic mouse D665 Ab. Bars show the mean fold inductions (F.I.) ± SD, after normalisation to GAPDH. The data are representative of three independent experiments. Statistical significance was calculated by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
