Journal:

Article Title: Evidence for transmembrane proton transfer in a dihaem-containing membrane protein complex

doi: 10.1038/sj.emboj.7601361

Figure Lengend Snippet: Catalytic activity (A), and monitoring of any generation of ΔpH (B) and ΔΨ (C) by wild type and E180Q-QFR. (A) Normalised, apparent progress curves of MMANH2 oxidation by fumarate as catalysed by wild-type QFR (blue trace) and by E180Q-QFR (red) and not catalysed by E180Q-QFR in the presence of the inhibitor DHN (20 μM, green). Proteoliposomes (200 μg) containing the respective QFR enzyme and MMANH2 (10 mM/mg phospholipid) were suspended in N2-flushed buffer. The reaction was started by the addition of fumarate (20 μM). The absorbance difference A280−A320 was monitored as a function of time. The traces were normalised by setting the absorption difference of A280−A320 to zero and the time of fumarate addition to zero. The progress curves were recorded in the same experiment as in B using a diode array spectrophotometer. (B) Normalised progress curves of the pyranine absorbance ratio offset as an indicator of the acidification of the proteoliposomal interior upon MMANH2 oxidation by fumarate. The colour-coding is as defined for (A). The absorbance ratio offset Δ(A450/A415) depended only on the H+ concentration in the liposomes and was monitored as a function of time. The reaction was started by the addition of fumarate with the time set to 0. The traces were normalised by setting the initial absorbance ratio of A450/A415 to zero. (C) Recording of the external TPB− concentration (logarithmic scale) in a suspension of proteoliposomes during fumarate reduction by MMANH2. To a solution containing proteoliposomes (200 μg in 50 mM HEPES, pH 7.5) with reconstituted wild-type QFR (blue trace) or E180Q-QFR (red trace), MMANH2 (10 mM/mg phospholipid), TPB− was added in 1 mM aliquots for calibration (open arrows pointing upwards). Reaction was started by the addition of 20 μM fumarate (arrows pointing downwards). The E180Q-QFR in the presence of 20 μM DHN (green trace, addition of DHN indicated by the pink arrow) did not generate any appreciable ΔΨ. The TPB− uptake was calculated from the amplitude between the first fumarate addition and the level where the TPB− concentration reached a minimum. The concentration inside the proteoliposomes was corrected according to Equation (1). Because the electrodes had different Nernst factors, the respective TPB− concentrations are indicated in the plot in the respective colour.

Article Snippet: For the acidification measurements, 10 mM pyranine (Molecular Probes, Leiden, NL) was added to the buffer.

Techniques: Activity Assay, Spectrophotometry, Concentration Assay, Liposomes, Suspension

Journal: Cancer Research

Article Title: Inhibition of Lymphotoxin-β Receptor–Mediated Cell Death by Survivin-ΔEx3

doi: 10.1158/0008-5472.can-05-2479

Figure Lengend Snippet: Figure 1. Interaction of caspase-3 with KSHV-K7 and survivin-DEx3. A, inhibition of caspase-3 activity in LIGHT/ IFN-g-mediated cell death. Wild-type Hep3BT2 (WT) and transfectants stably expressing survivin-DEx3, KSHV-K7, and Bcl-2 were incubated with IFN-g and LIGHT for 12 hours, and caspase-3 activities in cell lysates were determined using the fluorescent probe MCA-DEVD.APK (DNP). Columns, average of three independent experiments done in duplicate; bars, SD. *, P < 0.05, significantly different from untreated as determined by ANOVA. B, interaction between survivin-DEx3, KSHV-K7, and activated caspase-3. HEK293 cells were treated with TNF-a (10 ng/mL) plus cycloheximide (1 Ag/mL) for 8 hours, and cell lysates were then incubated with 1 Ag recombinant FLAG-KSHV-K7 or FLAG-survivin-DEx3 protein purified from pFLAG-KSHV- K7-transfected or pFLAG-survivin-DEx3- transfected HEK293T cells followed by immunoprecipitation using anti-FLAG mAb. The presence of caspase-3 in the immunoprecipitates was determined using an anti-caspase-3 (CPP32) mAb. C, inhibition of activated caspase-3. Recombinant activated caspase-3 was incubated with Z-DEVD or transfected cell lysates in the presence of MCA-DEVD.APK (DNP) for 1 hour at 37jC. Caspase-3 activities were determined using a fluorescence spectrophotometer.

Article Snippet: The blots were probed with mAbs against cytochrome c (6H2.B4; Imgenex, San Diego, CA), cytochrome oxidase IV (COX IV; Molecular Probes), actin (Sigma), and Smac/DIABLO (R&D Systems).

Techniques: Inhibition, Activity Assay, Stable Transfection, Expressing, Incubation, Recombinant, Purification, Transfection, Immunoprecipitation, Fluorescence, Spectrophotometry

Journal: Cancer Research

Article Title: Inhibition of Lymphotoxin-β Receptor–Mediated Cell Death by Survivin-ΔEx3

doi: 10.1158/0008-5472.can-05-2479

Figure Lengend Snippet: Figure 4. Survivin-DEx3 prevents the release of cytochrome c and Smac from Hep3BT2 cells after LIGHT/IFN-g treatment. A, mitochondrial integrity assay by subcellular fractionation. Cells were incubated with LIGHT/IFN-g for 20 hours followed by digitonin-based subcellular fractionation to separate the cell lysate into cytosol-enriched (left) and mitochondria-enriched (right) fractions and by Western blot analysis using antigen-specific antibodies. (Each fraction represents 5 106 cells.) Actin and COX IV served as references for the cytosol and mitochondria, respectively, as described in Materials and Methods. B, colocalization of survivin-DEx3 and Smac. Hep3BT2 cells were incubated with LIGHT/IFN-g for 20 hours. Survivin-DEx3 and KSHV-K7 were stained with anti- Myc tag, whereas cytochrome c (Cyt c) and Smac were stained with anti–cytochrome c and anti-Smac antibodies, respectively. Smac and anti-Myc signals were merged. The cell nucleus was stained with Hoechst 33342. C, association of survivin-DEx3 with Smac. Hep3BT2 cells were cotransfected with FLAG-tagged survivin-DEx3, KSHV-K7, or survivin in conjunction with HA-tagged Smac for 18 hours followed by incubation with LIGHT/IFN-g for another 20 hours. Cells were then harvested, and cell lysates were incubated with M2 anti-FLAG antibody for immunoprecipitation before blotting onto nitrocellulose membrane. Blots were probed with anti-FLAG or anti-HA mAb. D, translocation of survivin-DEx3 to mitochondria. Hep3BT2 cells were cotransfected pDsRed2-Mito and either survivin-DEx3 or KSHV-K7 for 18 hours followed by incubation with LIGHT/IFN-g for various time intervals. Survivin-DEx3 and KSHV-K7 were stained with anti-Myc tag antibody; the cell nucleus was stained with Hoechst 33342. Colocalization in the merged images of green and red channels. E, BIR domain is essential for the interaction between survivin-DEx3 and Smac. Hep3BT2 cells (1 106) were cotransfected with FLAG-tagged survivin-DEx3 (2 Ag/mL) or survivin-DEx3 (DBIR; 2 Ag/mL) in conjunction with HA-tagged Smac (2 Ag/mL) for 18 hours followed by incubation with LIGHT/IFN-g for another 20 hours. Cells were then harvested, and cell lysates were incubated with M2 anti-FLAG antibody for immunoprecipitation before blotting onto nitrocellulose membrane. Blots were probed with anti-FLAG or anti-HA mAb. F, colocalization of Smac with survivin-DEx3 but not survivin-DEx3 (DBIR). Hep3BT2 cells (3 105) were cotransfected with pDsRed2-Mito, HA-tagged Smac, in conjunction with Myc-tagged survivin-DEx3 or survivin-DEx3 (DBIR) for 18 hours followed by incubation with LIGHT/IFN-g for 20 hours. Cells were incubated with anti-Myc antibody and FITC-conjugated anti-mouse IgG subsequently to detect survivin-DEx3 and survivin-DEx3 (DBIR) or stained with anti-Smac antibody and Cy5-conjugated goat anti-rabbit antibody subsequently. Colocalization in the merged images of green [FITC for survivin-DEx3 or survivin-DEx3 (DBIR)], red (DsRed for mitochondria), and blue (Cy5 for Smac/DIABLO) channels. Mt, mitochondrial fraction.

Article Snippet: The blots were probed with mAbs against cytochrome c (6H2.B4; Imgenex, San Diego, CA), cytochrome oxidase IV (COX IV; Molecular Probes), actin (Sigma), and Smac/DIABLO (R&D Systems).

Techniques: Integrity Assay, Fractionation, Incubation, Western Blot, Staining, Immunoprecipitation, Membrane, Translocation Assay

Journal: Cancer Research

Article Title: Inhibition of Lymphotoxin-β Receptor–Mediated Cell Death by Survivin-ΔEx3

doi: 10.1158/0008-5472.can-05-2479

Figure Lengend Snippet: Figure 6. Translocation of survivin-DEx3 from nucleus to cytosol and colocalization of survivin-DEx3 with ASK1 after LIGHT/IFN-g treatment. A, translocation of survivin-DEx3 from nucleus to cytosol. Hep3BT2 cells were transfected with Myc-tagged survivin-DEx3 followed by treatment with LIGHT/IFN-g for various time intervals. Localization of survivin-DEx3 was determined by confocal microscopy using anti-HA (for ASK1) and Cy3-conjugated donkey anti-rat secondary antibody (red) subsequently, whereas survivin-DEx3 was detected by anti-Myc antibody and FITC-conjugated goat anti-mouse secondary antibody (green) subsequently. Merge, merged images from survivin-DEx3 and ASK1 staining. The cell nucleus was stained with Hoechst 33342. Bar, 8 Am. B, subcellular fractionation of survivin-DEx3 and ASK1. Hep3BT2 cells were transfected with Myc-tagged survivin-DEx3 and treated with LIGHT/IFN-g. Cytosolic and nuclear fractions were prepared as described in Materials and Methods. Western blots of the two fractions (nuclear and cytosol) were probed with anti-FLAG (for survivin-DEx3) or anti-HA (for ASK1) antibody. Anti-tubulin and anti–poly(ADP-ribose) polymerase (PARP) were used as controls for the cytoplasmic and nuclear fractions, respectively. C, association of survivin-DEx3 with ASK1. Hep3BT2 cells were cotransfected with FALG-survivin-DEx3 and HA-ASK1 for 18 hours and incubated with LIGHT/IFN-g. Following subcellular fractionation, the nuclear and cytoplasmic fractions were immunoprecipitated with antibody and blotted onto nitrocellulose membrane. The blot was probed with anti-FLAG and anti-HA antibody to detect the presence of survivin-DEx3 and ASK1, respectively. D, mapping of interaction domain of survivin-DEx3 with ASK-1. HeLa (1 106) cells were transfected with pCR3.1, pCR3.1-survivin-DEx3, or its deletion mutants for 18 hours followed by immunoprecipitation using anti-HA to pull down survivin-DEx3 and deletion mutants and incubated with anti-ASK-1 mAb (top). Alternatively, cell lysates were precipitated by anti-ASK-1 mAb followed by incubation with anti-HA mAb to detect survivin-DEx3 and its mutants (bottom). E, HeLa cells were transfected with pCR3.1, pCR3.1-survivin-DEx3, or pCR3.1-survivin-DEx3 (DNLS) for 18 hours followed by incubation with LIGHT-R228E (100 ng/mL) for various time intervals, and the ASK1 activities in the immunoprecipitates were determined by in vitro kinase assay. Nu, nuclear fraction.

Article Snippet: The blots were probed with mAbs against cytochrome c (6H2.B4; Imgenex, San Diego, CA), cytochrome oxidase IV (COX IV; Molecular Probes), actin (Sigma), and Smac/DIABLO (R&D Systems).

Techniques: Translocation Assay, Transfection, Confocal Microscopy, Staining, Fractionation, Western Blot, Incubation, Immunoprecipitation, Membrane, In Vitro, Kinase Assay