Figure 2
Figure 2. Disruption of AKAP450 function impairs the phosphorylation of different molecules associated with the activation of LFA-1. (A) Top blot: 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 a representative Western blot of phospho-PLC-γ1 (Y783) is shown. Total PLC-γ1 expression was detected as a loading control. Bottom blots: J77/SEE-pulsed Raji cell conjugates and CH7C17/HOM-2 conjugates expressing control siRNA (siNeg) or AKAP450 siRNA (siAKAP). Control and AKAP450 knockdown T cells were conjugated as in panel A, and representative Western blots of phospho-PLC-γ1 (Y783) are shown. Total PLC-γ1 expression was detected as a loading control. One of 4 representative experiments is shown. (B) Top blot: J77 expressing GFP (J77-GFP) or C-term AKAP450-GFP (J77-C-term) were conjugated as in panel A, and a representative Western blot of phospho-PKC-θ (T538) is shown. Total PKC-θ expression was detected as a loading control. Bottom blots: J77/SEE-pulsed Raji cell conjugates and CH7C17/HOM-2 conjugates expressing control siRNA (siNeg) or AKAP450 siRNA (siAKAP). Control and AKAP450 knockdown T cells were conjugated as in panel A, and representative Western blots of phospho-PKC-θ (T538) are shown. Total PKC-θ expression was detected as a loading control. One of 4 representative experiments is shown. (C) Conjugates were formed between J77 cells expressing GFP or C-term AKAP450-GFP and SEE-pulsed Raji B cells for the different times (5 minutes, top panel; 20 minutes, bottom panel), and were stained for PKC-θ (red). 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. The graph shows the quantification of the total PKC-θ accumulation for the times indicated at the IS. A total of 25 conjugates were counted for each time and condition. Data are the arithmetic mean ± SD of PKC-θ accumulation. *P < .05 compared with J77 cells transfected with GFP (Student t test).

Disruption of AKAP450 function impairs the phosphorylation of different molecules associated with the activation of LFA-1. (A) Top blot: 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 a representative Western blot of phospho-PLC-γ1 (Y783) is shown. Total PLC-γ1 expression was detected as a loading control. Bottom blots: J77/SEE-pulsed Raji cell conjugates and CH7C17/HOM-2 conjugates expressing control siRNA (siNeg) or AKAP450 siRNA (siAKAP). Control and AKAP450 knockdown T cells were conjugated as in panel A, and representative Western blots of phospho-PLC-γ1 (Y783) are shown. Total PLC-γ1 expression was detected as a loading control. One of 4 representative experiments is shown. (B) Top blot: J77 expressing GFP (J77-GFP) or C-term AKAP450-GFP (J77-C-term) were conjugated as in panel A, and a representative Western blot of phospho-PKC-θ (T538) is shown. Total PKC-θ expression was detected as a loading control. Bottom blots: J77/SEE-pulsed Raji cell conjugates and CH7C17/HOM-2 conjugates expressing control siRNA (siNeg) or AKAP450 siRNA (siAKAP). Control and AKAP450 knockdown T cells were conjugated as in panel A, and representative Western blots of phospho-PKC-θ (T538) are shown. Total PKC-θ expression was detected as a loading control. One of 4 representative experiments is shown. (C) Conjugates were formed between J77 cells expressing GFP or C-term AKAP450-GFP and SEE-pulsed Raji B cells for the different times (5 minutes, top panel; 20 minutes, bottom panel), and were stained for PKC-θ (red). 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. The graph shows the quantification of the total PKC-θ accumulation for the times indicated at the IS. A total of 25 conjugates were counted for each time and condition. Data are the arithmetic mean ± SD of PKC-θ accumulation. *P < .05 compared with J77 cells transfected with GFP (Student t test).

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