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Blood, Vol. 113, Issue 15, 3530-3541, April 9, 2009
Incomplete T-cell receptor–β peptides target the mitochondrion and induce apoptosis
Blood Shani et al.
113: 3530
Supplemental materials for: Shani et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 99.2 KB)
- Table S1. Summary of the primers used in the study (PDF, 20.6 KB)
- Figure S1. Cloning of the TCR iJC-TCRβ transcript (JPG, 133 KB)
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(A) iJC-TCRβ transcript containing the intronic and exonic regions of Jβ2.6 with an in-frame methionine in the 5′ intronic region was cloned from thymic cDNA, as well as from MBA-13 mesenchymal stem cell and MEF cDNAs. (B) Mesenchymal TCR mRNA nucleotide sequence and predicted protein sequence. The corresponding sequences of the cDNA clones from the 3 sources were identical.
- Figure S2. Single chain TCRβ proteins are not massively degraded and can be extracted only by high concentration of SDS (JPG, 42.1 KB)
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(A) 293T cells were transfected with Flag-iJC and at 48 hours post transfection cell lysates were prepared using the following lysis buffers: RIPA (150 mM NaCl, 50 mM Tris, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 1 mM EDTA, pH 7.5) (1), TENN buffer (150 mM NaCl, 50 mM Tris, 1% NP40, 5 mM EDTA, pH 8) (2), RIPA+10% glycerol (3), Brij-97 lysis buffer (150 mM NaCl, 50 mM Tris, 1% Brij-97, 7mM EDTA pH 7.5) (4) and SDS sample buffer (20% glycerol, 8% SDS, 0.25 M Tris 8% 2-ME, 0.04% bromophenol blue pH 6.8) (5). Similar amounts from each extract were analyzed by Western blotting using an anti-Flag antibody. (B) 293 cells were transfected with the indicated constructs and 48 hours later cell lysates were prepared using 1% SDS lysis buffer (20mM Tris, 1mM EDTA, 1% SDS pH 7.5). Samples were analyzed by Western blotting using an anti-GFP antibody.
- Figure S3. EGFP-iJC and EGFP-VDJC-KKKNS induce apoptosis (JPG, 112 KB)
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(A) 293 cells and (B) COS-7 cells were transfected with the indicated constructs and 48 hours later were labeled by the TUNEL method (Rhodamine staining) and counterstained with Hoechst for nuclear staining. All images were taken by fluorescence microscopy (×60 oil immersion objectives).
- Figure S4. The induction of apoptosis by EGFP-iJC and EGFP-VDJC-KKKNS involves the release of cytochrome c from mitochondria and the activation of caspases (JPG, 154 KB)
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(A) COS-7 cells were transfected with the indicated vectors (green). After 48 hours, the cells were stained with mouse anti-cytochrome c antibodies followed by anti-mouse Cy3 secondary antibodies (red). A significant percent of the cells had their cytochrome c spread throughout the cytoplasm (E–P) while others still had their cytochrome c confined in mitochondria (A–D). The cells were then stained with Hoechst for nuclear detection (blue), as indicated on the panels and subjected to fluorescence microscopy analysis (×60). (B) 293 cells were transfected with the indicated constructs. At 48 hours post transfection cells were subjected to a flow cytometry cell cycle analysis.
- Figure S5. Bax/Bak signaling contributes to iJC-TCRβ–induced apoptosis (JPG, 64.4 KB)
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MEF deficient for Bax−∕−Bak−∕− cell line and NIH3T3 cells were transfected with EGFP or EGFP-iJC. Forty-eight hours post transfection the cells were stained with propidium iodide and analyzed for their cell cycle distribution. a–d: Bax−∕−Bak−∕− cells, e–h: NIH3T3 cells.
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