Figure 2

Arl4d interferes with Akt phosphorylation in activated T cells. (A,B,D,F) naïve OT-1 CD8 T cells from wild type (A,B,D,F) and Arl4d^−/− OT-1 T cells (D,F) were co-cultured with antigen-loaded wild type DC, wild type LSEC or Pdl1^−/− LSEC as indicated. (A,B) At the indicated times T cells were analysed by western blot for expression of pAkt^S473 and total Akt. Shown are cropped images of the blot (full length blots are presented in Supplementary Figure S2). Bar graphs show normalised expression levels of pAkt. (C,E) A constitutive active Arl4d mutant (Q80L), a myristoylation-deficient Arl4d mutant (G2A) or an empty pEGFP vector were expressed in Jurkat T cells. (C) Jurkat cells were stimulated with anti-CD3 antibodies and analysed for expression of phosphorylated Akt (S473, T308) by flow cytometry in GFP⁺ Jurkat T cells (n = 5). (D) wt and Arl4d^−/− T cells stimulated by wild type LSEC for 48 h were stained for pAkt^S473 and pAkt^T308 Representative histograms and mean fluorescence intensity (MFI) of CD8⁺ cells are shown. (E,F) Survival of Jurkat cells (E) 24 h after transfection with Arl4d-mutant constructs or (F) wild type and Arl4d^−/− OT-1 T cells stimulated by wild type LSEC. Cells were stained with Propidium iodide or 7-AAD and Annexin V. Live cells were defined as being GFP^pos/CD8α^pos and AnnV^neg and PI/7-AAD^neg. The data shown are representative of 3 (A,B), 5 (C,D,E) and 2 (F) separate experiments. Data are shown as mean +/− s.e.m. Statistical significance was calculated using a one-way ANOVA or a Student’s t-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.