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. 2004 Jul 19;166(2):173–178. doi: 10.1083/jcb.200309044

Figure 5.

Figure 5.

Stable RNA interference of Dlg1 in Jurkat cells enhances NFAT activity. (A) Cells expressing shRNA were lysed in 1% Triton X-100 buffer and an equal amount protein from each cell line was resolved on a 7.5% SDS gel and blotted with antibodies recognizing Dlg1, actin, GSK, and Lck. (B) Activation of NFAT luciferase reporter in Jurkat cells stably transfected to express shRNA targeted against the Dlg1 SH3 domain (shRNA1) or GK domain (shRNA2). Cells were transfected with NFAT-luciferase reporter. 24 h after transfection, cells were stimulated with anti-CD3 (5 μg/ml) for 8 h and lysed for assay. (C) Activation of NFAT luciferase reporter in Jurkat cells stably transfected to express shRNA targeted against the Dlg1 SH3 domain (shRNA1) or GK domain (shRNA2). Pooled stable cells were transfected with NFAT-luciferase reporter construct. 20 h after transfection, cells were stimulated with Raji cells pulsed with SEE for 8 h and lysed for luciferase assay. (Bottom) Immunoblot analysis confirms shRNA suppression of Dlg1 expression. White lines indicate that intervening lanes have been spliced out. Error bars represent SEM.