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Results: 3

1.
Figure 1

Figure 1. From: CD27, a member of the tumor necrosis factor receptor family, induces apoptosis and binds to Siva, a proapoptotic protein.

Ligation of CD27 induces apoptosis. (A) Ramos cells (0.5 × 106/ml) were cultured with increasing concentrations (0.1, 0.2, and 0.5 × 106/ml) of mock (lanes 2–4) and CD70 transfectants (lanes 5–7) for 2 days. Ramos, mock, and CD70 transfectants when cultured alone are shown (lanes 8–10). (B–E) Experiments with formaldehyde-fixed CD70 and mock transfectants, using Ramos, Raji, human CD27 transfected mouse pre-B 300 cell lines and human peripheral blood lymphocytes preactivated with phytohemagglutinin/IL-2. The effect of coligation of surface IgM (Ramos and Raji) and Ig (murine pre-B 300) using anti-human IgM and anti-mouse IgG antibodies (10 μg/ml), respectively, on CD27-induced apoptosis is also shown (B, lanes 7 and 4; C, lane 6 vs. lane 3; and D, lane 5 vs. lane 3).

K. V. S. Prasad, et al. Proc Natl Acad Sci U S A. 1997 June 10;94(12):6346-6351.
2.
Figure 2

Figure 2. From: CD27, a member of the tumor necrosis factor receptor family, induces apoptosis and binds to Siva, a proapoptotic protein.

(A) Siva cDNA sequence obtained from HeLa and human thymus cDNA libraries with the translated ORF (189 aa) is shown. The initiation codon is defined by the upstream in-frame stop codon (−42). The coding region is followed by a stop codon (568), a polyadenylylation signal (709–714). The sequence has been deposited in the GenBank under the accession number U2938. (B) A death domain (DD) homology region toward the amino terminus has been identified in Siva, and its homology to FADD and RIP DDs was determined with use of the multiple sequence alignment program clustal. The shaded regions show homology and the residues in boldface type depict identical residues conserved among the three regions. (C) Putative B-box domain of Siva. Homologous residues between Siva and other proteins in the B-box domain are shaded.

K. V. S. Prasad, et al. Proc Natl Acad Sci U S A. 1997 June 10;94(12):6346-6351.
3.
Figure 3

Figure 3. From: CD27, a member of the tumor necrosis factor receptor family, induces apoptosis and binds to Siva, a proapoptotic protein.

(A) mRNA expression of Siva by Northern blot analysis is shown (upper bands) and is compared with the expression of G3PDH (lower bands). (B) CD27 binds to Siva in 293 cells. CD27WT DNA was transiently coexpressed with either GFP or GFP–Siva for 2 days. The CD27 receptor expression appeared to be similar as seen by flow cytometric analysis (data not shown). CD27 immunoprecipitates using anti-CD27 antibody (IA4) were separated by SDS/PAGE, transferred, and blotted with anti-GFP antiserum. Similar amounts of GFP and GFP–Siva were expressed as seen in the lanes corresponding to whole-cell lysates (lanes 4 and 3). CD27 coprecipitaed GFP–Siva (lane 1) but not GFP (lane 2). (C) Siva-induced apoptosis. Jurkat, Raji, SKW, Ramos, and 293 cells were transiently transfected (2 days) with GFP (hatched bar) and GFP–Siva (solid bar). The histograms represent the percentage of dead cells (based on cell morphology) in the cells expressing green fluorescence. In the case of 293 cells, along with GFP and GFP–Siva, pSVb containing the β-galactosidase gene was cotransfected. β-Galactosidase expression was visualized by X-Gal. The percentage of rounded shrunken cells in the fluorescent population of cells is presented. (D) Transient expression of GFP–Siva but not GFP can induce apoptosis in 293 cells (lane 3 vs. lane 2), the murine pre-B cell line (lane 6 vs. lane 5), and Raji cells (lane 8 vs. lane 7), as judged by the generation of DNA ladder.

K. V. S. Prasad, et al. Proc Natl Acad Sci U S A. 1997 June 10;94(12):6346-6351.

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