FIG. 2.

CaMKII redistributes to the immune synapse in an activation-dependent manner. (A) Raji cells were loaded with a fluorescent cell tracker chloromethylbenzoyl derivative of aminocoumarin (blue) and pulsed with SEE for 20 min. See Materials and Methods for details. Alexa Fluor 488 is shown in green, and phalloidin conjugated with Alexa Fluor 568 is shown in red. Representative results from one of three experiments are shown. (B) Quantification of CaMKII redistribution to the contact area in Jurkat cells. Conjugates displaying CaMKII redistribution to the submembranous area were counted under the fluorescence microscope at a magnification of ×100. Results are representative of three independent experiments. n, total number of conjugates examined for each experimental condition. (C) After incubation with KN93 or DMSO, Jurkat cells were stimulated with anti-CD3 antibody or TNF-α and then the nuclear extract was obtained as described in Materials and Methods. The nuclear extract (10 μg of total proteins) was subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and blotted with anti-p65 antibody or antiretinoblastoma (Rb) antibody.