Overexpression of C-term AKAP450-GFP impairs the activation of the TCR/CD3 complex and T-cell signaling molecules. (A) Conjugates were formed between J77 cells expressing GFP or C-term AKAP450-GFP and SEE-pulsed Raji B cells, and were stained for CD3ζ (red) and activated CD3ϵ (APA1/1: magenta). Yellow arrows indicate the positions of immune synapses; blue arrow, position of the mislocalized MTOC in cells overexpressing C-term AKAP450-GFP. Asterisks in DIC images identify CMAC-loaded Raji APCs. Scale bars represent 10 μm. (B) Histogram shows quantification of the relation between APA1/1 signal and CD3 signal in conjugates formed as in panel A. A total of 50 conjugates were counted for each condition. Results are the arithmetic mean ± SD. *P < .05 compared with J77 cells transfected with GFP control (Student t test). (C) Representative Western analysis of phospho-CD3ζ (Y83) in J77/SEE-pulsed Raji cell conjugates as in panel A, formed for the indicated times. Total CD3 expression was detected as a loading control. One of 4 representative experiments is shown. (D) J77 expressing GFP (J77-GFP) or C-term AKAP450-GFP (J77-C-term) were conjugated with SEE-pulsed Raji cell for the times indicated, and cell proteins were separated by SDS-PAGE and processed for Western blotting. Blots show the phosphorylation of specific sites on Vav1 (Y174) and LAT (Y191, Y132) together with the total expression of these proteins. Arrows indicate target band sizes. One of 4 experiments is shown.