We next analyzed TCR-mediated activation of NF-AT and NF-kB transcription factors. siRNA-treated Jurkat T cells were cotransfected with firefly luciferase constructs driven by NF-AT or NF-kB binding sequences and with a renilla luciferase control. PALS1 knockdown had only a marginal effect on NF-AT activity following stimulation with PMA and ionomycin, or with antibodies to CD3 and CD28 ( Figure 3C and Figure S2 ). In sharp contrast, NF-kB activity was significantly reduced without PALS1 ( Figure 3D ). Interestingly, tumor necrosis factor-a (TNFa)induced NF-kB activation remained essentially unaffected, underscoring the selective involvement of PALS1 in the TCR-NF-kB pathway ( Figure S3 ). Altogether, our data unveiled an unexpected role for PALS1 in TCR-mediated NF-kB activation.