Skip to main content
. 2007 Sep 20;26(20):4368–4379. doi: 10.1038/sj.emboj.7601845

Figure 1.

Figure 1

SiRNA-mediated knockdown of OGT impairs early T-cell activation. (A) Jurkat cells were transfected with a negative control siRNA (Ctrl) or siRNAs specific for Lck or OGT (OGT_1). After transfection, the cells were transferred to fresh culture medium for 24, 48 and 72 h before stimulation with plate-bound anti-CD3 and anti-CD28 antibodies for 5.5 h and RNA was isolated. IL-2 mRNA as well as mRNAs for Lck or OGT were quantified by real-time PCR as described in Materials and methods. Experiments were performed in triplicates. Expression is given as the percentile of the amount of specific mRNA (black bars) compared to the amount in cells transfected with Ctrl (gray bars). (B) Jurkat cells were transfected as described in (A) and incubated for 24, 48 or 72 h prior lysis. OGT immunoprecipitations were performed and analyzed by Western blot using an antibody to OGT (TI-14). (C) Jurkat cells stably expressing a luciferase reporter plasmid under the control of the IL-2 promoter were transfected with a negative Ctrl or siRNAs specific for Lck or OGT (OGT_1, OGT_2, OGT_3). After transfection, the cells were transferred to fresh culture medium for 24 h before stimulation with plate-bound anti-CD3 and anti-28 antibodies for 5.5 h. Luciferase activity was quantified as described in Materials and methods. Experiments were performed in triplicates. Luciferase activity of the control transfection using nonsense siRNA is set to 100% and the luciferase activities after OGT and Lck downmodulation are given as percentage of the control. (D) Jurkat cells were treated as described in (C) and costimulated with plate-bound anti-CD3/28 antibodies for 16 h. After stimulation, CD69 surface expression was measured by Flow cytometry as described in Materials and methods. Experiments were performed in triplicates. For CD69 externalization, the control transfection using nonsense siRNA is set to 100% and the CD69 externalization after OGT and Lck downmodulation is given as percentage of the control. (E) Experiments were performed as described in panel (D), but cells were stimulated with PMA/Ionomycin. For CD69 externalization, the control transfection using nonsense siRNA is set to 100% and the CD69 externalization after OGT and Lck downmodulation is given as percentage of the control.