Figure 7

Co-localization of TRAF2 and TNFR2 following cytokine treatment is prolonged in NOD β cells. Expression of the adaptor molecules TRAF2 and RIP in islets. (A) Immunohistochemical staining of pancreas sections from 12–14 week old NOD and Idd9 congenic mice with anti-TRAF2 antibody. Immunoblotting with antibodies to TRAF2 (B) and RIP (C) of protein lysates prepared from 5 week old Idd9 congenic (I) and NOD (N) islets stimulated for 3 days +/- 1,000 U/ml TNF + 1,000 U/ml IFNγ. In each case, membranes were stripped and re-blotted with antibodies to actin as loading control. Results representative of 2–3 independent experiments. (D) Co-localization of TRAF2 and TNFR2 following cytokine treatment determined by confocal imaging. Representative images of β cells from 5 week old NOD and Idd9 congenic mice treated with 1,000 U/ml IFNg + 1,000 U/ml TNF for 0, 30 and 60 minutes, stained with antibodies to TNFR2, TRAF2 and insulin, and the nuclear dye DAPI. The merged image of TNFR2 (green), TRAF2 (red) and DAPI (blue) is also shown. Dead cells were excluded using an EtBr-derived dye. In (E) the average TNFR2/TRAF2 colocalization coefficient (M1) +/- SEM is shown for each time point. TNFR2 positive cells are rare within the islet population, but on average 18 TNFR2 positive cells were analyzed per treatment. The threshold for TNFR2 and TRAF2 staining was determined by staining an aliquot of the cells in parallel minus each primary antibody.