Loss of IRF-4–binding protein leads to the spontaneous development of systemic autoimmunity
J. Clin. Invest. Jessica C. Fanzo, et al. 116:703 doi:10.1172/JCI24096 [
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Figure 7Defective ERK1/2 activation in
IBPtrap/trap T cells. (
A) Lck and ZAP-70 activation in
IBPtrap/trap T cells was detected by Western blotting utilizing antibodies specific for Tyr 394 phosphorylated Lck (upper panel) and Tyr 319 phosphorylated ZAP-70 (middle panel). Reprobing with an antibody against total Lck is shown as a loading control (lower panel). (
B) TCR-mediated calcium mobilization in
IBP+/+ and
IBPtrap/trap T cells. Lymph node cells were loaded with Fura-red and Fluo-4 and surface stained with APC-labeled anti-CD4 antibody. Cells were then precoated with 5 μg anti-CD3ε (2C11) antibody and cross-linked with goat anti-hamster Ig. Histogram data are presented as a median ratio of calcium mobilization gated on CD4
+ cells as measured by FACS. The black line represents
IBP+/+ T cells, and the gray line represents
IBPtrap/trap T cells. Arrow indicates the addition of cross-linking antibody. (
C) ERK activation in
IBP+/+ and
IBPtrap/trap T cells. Cells were stimulated with anti-CD3ε antibody for the indicated times or PMA (50 ng/ml) for 2 minutes as a control. Whole-cell lysates were prepared and active ERK1/2 detected by Western blotting using an anti–phosphorylated ERK antibody (upper panel). Total ERK1/2 levels are shown in the lower panel. (
D) Induction of c-Fos in
IBPtrap/trap T cells. Primed T cells from
IBP+/+ and
IBPtrap/trap mice were stimulated with anti-CD3ε antibody (5 μg/ml) and anti-CD28 antibody (5 μg/ml) for 0, 1, or 2 hours. Lysates were prepared and levels of c-Fos detected by Western blotting using an anti–c-Fos antibody (upper panel). Reprobing with a β-actin antibody is shown as a loading control (lower panel).
