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Blood, Vol. 111, Issue 7, 3653-3664, April 1, 2008
Antigen-induced clustering of surface CD38 and recruitment of intracellular CD38 to the immunologic synapse
Blood Muñoz et al.
111: 3653
Supplemental materials for: Muñoz et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 65.1 KB)
- Figure S1. Effect of anti-CD38 mAb on antigen-induced early signaling events, and on translocation of PKCθ to the IS (JPG, 129 KB)
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(A) Western-blot analysis with the anti-phospho LAT (Tyr191), followed by anti- -actin for protein loading-control of cell lysates of CH7C17 T cells incubated with unpulsed (−HA), or HA-pulsed (+HA) HOM-2 B cells for the period of times indicated. The CH7C17 T cells were either preincubated with vehicle (−IB6), or preincubated for 30 min at 37°C with the anti-CD38 mAb IB6 (+IB6), and washed before mixing them with the HOM-2 B cells. The picture is representative of three independent experiments. (B) Densitometric analysis of the blot in panel A corrected by protein-loading control (pLAT:Actin ratio ×100). (■) CH7C17 T cells incubated with unpulsed HOM-2 cells for the indicated periods of time, (□) CH7C17 T cell preincubated with anti-CD38 mAb IB6 and then, with unpulsed HOM-2 cells, (●) CH7C17 cells incubated with HA-pulsed HOM-2 cells, (○) CH7C17 cells preincubated with IB6 and then incubated with HA-pulsed HOM-2 cells. (C) Western-blot analysis with anti-phosphoErk (Thr202/Tyr204), followed by anti--actin for protein-loading control. (D) Densitometric analysis of the blot in panel C was corrected by protein-loading control. Symbols as in panel B. (E) HOM-2 B cells were labeled with CMAC blue and incubated in the presence of HA peptide. HA-pulsed HOM-2 and CH7C17 T cells were mixed at 1:1 ratio, briefly centrifuged and incubated at 37°C for 5 min before resuspension and extension on coverslips were they were incubated for another 20 min at 37°C. Cells were then fixed, permeabilized and double stained for CD3- with the anti-CD3- mAb 1D4.1 (green), and for PKC with the polyclonal rabbit antiserum #2059 (red). Cells were examined using an Olympus Cell R IX81 motorized system inverted microscope. Original magnification ×100. Bar 4 µm. (F) CH7C17 T cells were preincubated with the anti-CD38 mAb IB6 for 30 min at 37°C, then washed and mixed with HA-pulsed HOM-2 cells and processed as in E. (G) Comparative analysis of the number of conjugates with PKC at the IS relative to the conjugates with a mature IS (CD3-+ at the IS) in the absence of IB6 preincubation (filled bars), or preincubated with IB6 (open bars).
- Video 1. CD38 relocation to the immune synapse (MOV, 1.23 MB)
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J77 T cells transfected with CD38-GFP were adhered to fibronectin coated coverslips and mounted in chambers. Raji B cells preloaded with SEE and labelled in red with the probe CM-TMR were added to the chambers, and live J77 T cells were monitored using time-lapse confocal microscopy. Images were taken at 30s intervals. Video represents the superimposed images from GFP fluorescent signal (green) and the red fluorescence signal from B cells.
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