(a) Jurkat cells expressing either nonspecific (NS) or Sam68 specific siRNA were stimulated with anti-CD3/CD28 (1 µg/ml each, top panel) or 50 ng/ml PMA plus 1.5 µM ionomycin (PMA/I, bottom panel) for indicated periods. Whole cell lysates were extracted and immunoblotted (IB) for IκBα and Sam68, with β-actin as a loading control. (b) Immunoblots using indicated antibodies of cytosolic and nuclear fractions derived from Jurkat cells transfected with indicated siRNAs and left unstimulated or stimulated with PMA/I for indicated periods. PKCθ and PARP served as cytosolic and nuclear markers, respectively. (c) Confocal micrographs of Jurkat cells silenced and stimulated as in (b). The fixed cells were stained for Sam68, p65, and nuclei. (d) Percentage (mean ± S.D., n = 3) of Jurkat cells (n > 200) with nuclear p65 with nonspecific or Sam68 specific siRNA expression was quantified.