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. 2003 Jan 6;197(1):129–135. doi: 10.1084/jem.20021646

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Copyright © 2003, The Rockefeller University Press

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Figure 2. — CTLA-4 knockout T cells have more TCR in the raft than their littermate T cells. (A) CTLA-4 knockout and heterozygote littermate T cells were activated by plate-bound anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 2 d and rested for 1 d. 10⁶ cells were stained using different concentrations of cholera toxin FITC (8 μg/ml and 1 μg/ml) and analyzed using a flow cytometry. MFI, mean fluorescent intensity. (B) 10 mg of each lysate from CTLA-4 knockout and littermate T cells, which have been activated with anti-CD3 plus anti-CD28 as described for panel A, were subjected to sucrose density gradient separation. The gradient was divided into six fractions that were analyzed for Fyn (1st panel) and cholera toxin horseradish peroxidase. TCRζ was IP from each fraction and analyzed by WB using anti-ptyr antibody (3rd panel). The membrane was stripped and reprobed using an anti-TCRζ antibody. The 4th and 5th panels depict short (1 min) and long (10 min) exposure of the membrane, respectively. (C) The band intensity of the phosphorylated TCRζ in the raft fraction from above experiments was measured by densitometry. The intensity of TCRζ in CTLA-4 knockout T cells is shown as the fold increase as compared with littermate T cells.

Figure 2. — CTLA-4 knockout T cells have more TCR in the raft than their littermate T cells. (A) CTLA-4 knockout and heterozygote littermate T cells were activated by plate-bound anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 2 d and rested for 1 d. 10⁶ cells were stained using different concentrations of cholera toxin FITC (8 μg/ml and 1 μg/ml) and analyzed using a flow cytometry. MFI, mean fluorescent intensity. (B) 10 mg of each lysate from CTLA-4 knockout and littermate T cells, which have been activated with anti-CD3 plus anti-CD28 as described for panel A, were subjected to sucrose density gradient separation. The gradient was divided into six fractions that were analyzed for Fyn (1st panel) and cholera toxin horseradish peroxidase. TCRζ was IP from each fraction and analyzed by WB using anti-ptyr antibody (3rd panel). The membrane was stripped and reprobed using an anti-TCRζ antibody. The 4th and 5th panels depict short (1 min) and long (10 min) exposure of the membrane, respectively. (C) The band intensity of the phosphorylated TCRζ in the raft fraction from above experiments was measured by densitometry. The intensity of TCRζ in CTLA-4 knockout T cells is shown as the fold increase as compared with littermate T cells.