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Blood, Vol. 114, Issue 3, 572-579, July 16, 2009
A single nucleotide polymorphism determines protein isoform production of the human c-FLIP protein
Blood Ueffing et al.
114: 572
Supplemental materials for: Ueffing et al
Western blot analysis For Western blot analysis cells were lysed in TPNE buffer (PBS adjusted to 300 mM NaCl, 1% Triton-X100, 2 mM EDTA, 1 mM PMSF and 1 µg/ml each of leupeptin, aprotinin, chymostatin and pepstatin A). 20 µg of postnuclear supernatant protein as determined by the BCA method (Pierce Biotechnology, Rockford, IL) were separated by 12% SDS-PAGE, blotted onto a nitrocellulose membrane (Amersham, Freiburg, Germany) and blocked with 5% non-fat dry milk in PBS/Tween (0.05% Tween-20 in PBS). After washing with PBS/Tween the blots were incubated overnight with specific antibodies at 4°C. Blots were washed again with PBS/Tween, incubated with horse radish peroxidase-coupled secondary antibodies (1:20,000) for 1 h at room temperature, washed again and developed with a chemiluminescence reagent (Amersham). For stripping, blots were incubated in Re-Blot mild solution (Chemikon, Hofheim, Germany) according to manufacturer's procedure. Quantification of protein expression was carried out either using a LAS 3000 CCD camera (FUJIFILM, Düsseldorf, Germany) and AIDA software (Raytest, Sprockhövel, Germany), or after scanning of the blots by densitometric analysis employing the ImageJ software 1.39u.1 Immunoprecipitations To analyze ubiquitination of c-FLIPR and c-FLIPS, HEK293T cells were transiently transfected with FLAG-tagged ubiquitin with or without full-length or truncated c-FLIPR or c-FLIPS using Lipofectamine 2000 transfection reagent. 20 h later, cells were incubated with 10 µg/ml MG-132 for further 4 h. Then, cells were lysed in 20 mM Tris/HCl, pH 7.4, 1% Triton X-100, 10% glycerol, 150 mM NaCl, 1 mM PMSF and 1 µg/ml of leupeptin, aprotinin, chymostatin and pepstatin A for 15 min on ice and centrifuged (15 min, 14,000 g). Subsequently, immunoprecipitations were performed with 2 µg anti-GFP coupled to protein G beads (Sigma) for 4 h at 4°C. Finally, beads were washed three times with 750 µl ice-cold DISC buffer and analyzed by Western blotting. REFERENCE 1. Abramoff MD, Magelhaes PJ, Ram SJ. Image processing with ImageJ. Biophotonics Int. 2004;11:36–42.
Files in this Data Supplement:
- Table S1. Ape and human primary cells and cell lines analyzed for c-FLIPS and c-FLIPR isoform expression (PDF, 38.7 KB)
- Table S2. Percentages of male and female control and follicular lymphoma samples (PDF, 16.3 KB)
- Table S3. Multivariate analysis considering sex and association with rs10190751 A (PDF, 26.7 KB)
- Figure S1. Quantification of c-FLIPS and c-FLIPR protein levels as shown in Fig. 4 (JPG, 92.2 KB)
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(A) Quantification of the Western blot shown in Fig. 4A, in which L428 cells were treated for the indicated times cycloheximide. The exposed film was scanned and the corresponding TIFF file was analyzed using ImageJ software 1.39u. (B) Top: Western blot showing 293T cells transiently transfected with the indicated cDNA amounts for c-FLIPS or c-FLIPR. 24 h post transfection cells were treated with cycloheximide. Below: Quantification of c-FLIPS and c-FLIPR protein expression carried out using a LAS 3000 CCD camera (FUJIFILM, Düsseldorf, Germany) and AIDA software (Raytest, Sprockhövel, Germany).
- Figure S2. Protein stability of c-FLIPS and c-FLIPR in Boe and L1236 cells (JPG, 52.3 KB)
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(A) Boe cells were treated for the indicated times with 10 µg/ml cycloheximide. c-FLIP expression was analyzed by Western blot. Tubulin served as a loading control. (B) L1236 cells were treated for the indicated times with 10 µg/ml cycloheximide. c-FLIP expression was analyzed by Western blot. β-actin served as a loading control.
- Figure S3. mRNA stability of c-FLIPS and c-FLIPR (JPG, 53.1 KB)
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(A) L428 cells were treated for the indicated times with α-amanitin (2 µg/ml). Endogenous c-FLIP isoform mRNA stability was analyzed by RT-PCR. β-actin served as loading control. (B) 293T cells were cotransfected with equal amounts of c-FLIPS-and c-FLIPR-encoding plasmids. 24 h later, cells were stimulated for the indicated times with α-amanitin (2 µg/ml). mRNA stability was assessed by RT-PCR. The asterisk marks a band of unclear identity that appears specifically in the transfected cells. β-actin served as loading control.
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