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. 2002 May;160(5):1647–1654. doi: 10.1016/S0002-9440(10)61112-1

Figure 2.

Figure 2.

Blocking CD30 signaling by dominant-negative TRAF proteins in H-RS cell lines. A: Left, aggregation of TRAF2 and TRAF5 in HEK293 cells highly expressing CD30. TRAF2 and TRAF5 were aggregated in HEK293 cells highly expressing CD30 and retaining constitutive activation of NF-κB (293CD30), but not in HEK293 cells with an empty vector (293vec). Immunofluorescence studies were done as described in Figure 1, A and B ▶ . Right, immunoblot analysis of TRAF2 and TRAF5. The levels of TRAF protein expression are almost the same between 293CD30 and 293vec cells. B: Left, electrophoretic mobility shift analysis (EMSA) with an NF-κB probe. H-RS cell derived cell lines show constitutive activation of NF-κB. Jurkat cells treated with TNF-α were used as a positive control. Right, down-regulation of basal activities of NF-κB-driven luciferase by dominant TRAF proteins in H-RS cell lines, but not in Jurkat cells. Luciferase activities are expressed as relative levels of triplicated experiments where those co-transfected with a vacant expression vector are expressed as 100%. Transfection efficiencies were corrected by dual luciferase assays using pRL-TK-Luc. C: Expression of a dominant-negative TRAF2 abrogates cytoplasmic aggregation of endogenous TRAF proteins in L-428 and L-540 cells. Transfected ΔTRAF2 was detected by the C-terminal anti-TRAF2 mouse monoclonal antibody (Santa Cruz) and the endogenous TRAF2 by N-terminal anti-TRAF2 rabbit antibody (Santa Cruz). Secondary antibodies used are Texas Red-labeled anti-mouse immunoglobulin sheep antibody and FITC-labeled anti-rabbit immunoglobulin donkey antibody (both from Amersham Pharmacia Biotech). Strong staining of the highly expressed ΔTRAF2 protein by the C-terminal antibody made it possible to discriminate it from the endogenous TRAF2 protein. In the left panel, figures show the results with anti-N-terminal TRAF2 (N-19) (α-N-TRAF2) rabbit antibody and anti-C-terminal TRAF2 (H-10) (α-C-TRAF2) mouse monoclonal antibody (both from Santa Cruz). Arrows indicate cells that express transduced ΔTRAF2. In the right panel, merged figures of immunofluorescence by anti-N-terminal and anti-C-terminal TRAF2 antibodies. Secondary antibodies used are: FITC-labeled anti-rabbit donkey antibody and Texas Red-labeled anti-mouse immunoglobulin sheep antibody (both from Amersham Pharmacia Biotech). Original magnification, ×400.