Journal: PLoS ONE

Article Title: Drosophila Signal Peptidase Complex Member Spase12 Is Required for Development and Cell Differentiation

doi: 10.1371/journal.pone.0060908

Figure Lengend Snippet: yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B (ey-flp; 82B) control (A–A'') has limited Caspase expression and normal Elav expression compared to yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12 C24 (ey-flp; spase12 C24 ) (B–B'') mosaic discs in which Caspase is upregulated and Elav expression strongly disrupted.

Article Snippet: The following primary antibodies were used: rabbit anti-GFP (1∶1000, Molecular Probes), mouse anti-GFP (1∶1000, Molecular Probes), rabbit anti-Caspase (Cell Signaling), mouse anti-Arm (1∶500, DSHB) , and rat anti-Elav (1∶500, DSHB) .

Techniques: Expressing

Journal: PLoS ONE

Article Title: A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

doi: 10.1371/journal.pone.0027680

Figure Lengend Snippet: A: 100 µM VEID-Afc was incubated with different amounts of caspase -3, -6 or -7 for 1 h at 37°C. Fluorescence generated by cleavage was monitored over time, and the initial, linear portion of the curve was used to calculate the reaction velocity. VEID is preferentially cleaved by caspase-6, but also cross-reacts with caspases -3 and -7 at higher concentrations. Error bars are the SEM of N≥3 of 3 independent experiments. B: 5 µM VEID-Afc was incubated with 0.5 µM caspase-6 or different amounts of caspase-3 for 1 h at 37°C. The reaction velocity was calculated as in A. Even at this low concentration of VEID-Afc, the peptide substrate can be cleaved by caspase-3, and an 8 fold higher molar concentration of caspase-3 than caspase-6 results in a higher signal for VEID cleavage by caspase-3 than caspase-6. Error bars are the SEM of N = 3 independent experiments, statistical significance was assessed by 1-way ANOVA and post-hoc Dunnett comparisons: *** p<0.0001. C: Pure lamin A protein was incubated with different amounts of caspase -3, -6 or -7 for 30 min at 37°C. Samples were separated by SDS-PAGE and both fragments of cleaved lamin A was detected by Western blotting with antibodies #2031 (full-length lamin A and N-terminal fragment) and #2032 (C-terminal fragment). No cleavage was observed with caspases -3 or -7, while caspase-6 generated lamin A fragments in a dose-dependent manner. A representative image of 3 independent experiments is shown.

Article Snippet: Pure lamin A protein and lamin B1 antibody were from Abcam (cat. nos. ab8982 and ab83472), antibody against full-length caspase-6 was from Cell Signaling Technology (cat. no. 9762), antibody against caspase-3 was from Cell Signaling Technology (cat. no. 9662), antibody against actin was from Chemicon (cat. no. MAB1501R).

Techniques: Incubation, Fluorescence, Generated, Concentration Assay, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

doi: 10.1371/journal.pone.0027680

Figure Lengend Snippet: A: Caspase-6 protein (full-length, 32 kDa) is detected in MEFs generated from C6wt, but not C6ko mice. B: Endogenous lamin A protein (70 kDa) is cleaved in wt, but not C6ko MEFs after staurosporine stress for 4 h or longer. The antibody cross-reacts with full-length lamin C (60 kDa), and the cleaved band at 28 kDa has the same size for both lamin A+C (lower panel). C: MCF-7 cells express caspase-6, but not caspase-3 protein, whereas both C6wt and C6ko MEFs contain both caspases. hu: human, m: mouse, ns: non-specific band. D: MCF-7 cells were stressed with 5 µM camptothecin for different amounts of time and the cleavage of endogenous lamin A and C proteins was monitored by Western blotting with antibodies antibodies #2031 (full-length lamin A+C) and #2032 (C-terminal fragments). E: Schematic representation of lamin A and C and the caspase-6 cleavage site at AA 230. The N-terminal fragments generated by caspase-6 cleavage (red) have the same size (28 kDa) for both lamin A+C. F: C6wt or C6ko MEFs were stressed with 50 nM staurosporine for different amounts of time, lysates were generated and analyzed for cleavage of VEID-Afc. C6wt cells show a significant increase in fluorescence at each timepoint, the fluorescence signal obtained from C6ko lysates only reach a statistically significant difference from baseline after 4 h. C6ko MEFs stressed with staurosporine for 4 h or more show the same levels of VEID proteolysis as wt cells. Error bars are the SEM of N = 3 of 4 independent experiments. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p<0.0001, ** p<0.001, * p<0.01.

Article Snippet: Pure lamin A protein and lamin B1 antibody were from Abcam (cat. nos. ab8982 and ab83472), antibody against full-length caspase-6 was from Cell Signaling Technology (cat. no. 9762), antibody against caspase-3 was from Cell Signaling Technology (cat. no. 9662), antibody against actin was from Chemicon (cat. no. MAB1501R).

Techniques: Generated, Western Blot, Fluorescence

Journal: PLoS ONE

Article Title: A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

doi: 10.1371/journal.pone.0027680

Figure Lengend Snippet: A+B: Quantities of down to 10 nM active caspase-6 can be detected with the novel lamin A-based caspase-6 assay while maintaining a signal-noise ratio of >3. C: The peptide inhibitor VEID-CHO shows a similar IC 50 value in both the VEID- and the lamin A- based caspase-6 assays. D: The lamin A-based caspase-6 assay is highly specific, with no signal generated from C6ko cells even after 6 h of staurosporine stress. Error bars are the SEM of N≥3 of 3 independent experiments. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p<0.0001, ** p<0.001.

Article Snippet: Pure lamin A protein and lamin B1 antibody were from Abcam (cat. nos. ab8982 and ab83472), antibody against full-length caspase-6 was from Cell Signaling Technology (cat. no. 9762), antibody against caspase-3 was from Cell Signaling Technology (cat. no. 9662), antibody against actin was from Chemicon (cat. no. MAB1501R).

Techniques: Generated

Journal: PLoS ONE

Article Title: A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

doi: 10.1371/journal.pone.0027680

Figure Lengend Snippet: A: COS7 cells were transiently transfected with recombinant human caspase-6 and cultured for the indicated amounts of time. The active caspase-6 fragment can be observed by Western blotting 9 h after transfection and is increasingly abundant after 24 h. B: COS7 cells were transfected as in A . The cleavage of endogenous lamin A protein can be observed with the ELISA method after 24 h, whereas spiking of lysates with lamin A protein leads to a robust detection of caspase-6 activity 9 h after transfection. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p<0.0001.

Article Snippet: Pure lamin A protein and lamin B1 antibody were from Abcam (cat. nos. ab8982 and ab83472), antibody against full-length caspase-6 was from Cell Signaling Technology (cat. no. 9762), antibody against caspase-3 was from Cell Signaling Technology (cat. no. 9662), antibody against actin was from Chemicon (cat. no. MAB1501R).

Techniques: Transfection, Recombinant, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: PLoS ONE

Article Title: Necrosis Is the Dominant Cell Death Pathway in Uropathogenic Escherichia coli Elicited Epididymo-Orchitis and Is Responsible for Damage of Rat Testis

doi: 10.1371/journal.pone.0052919

Figure Lengend Snippet: Total testis protein (20 µg) from four different animals in each group were separated on 15% SDS-PAGE. Immunoblots were probed with anti-caspase-8 ( A ), anti-caspase-3 (B, upper panel) , anti-caspase-6 (B, lower panel) and anti-caspase-1 ( C ) antibodies and detected using chemiluminescence. RAW 264.7 cells treated with sodium nitroprusside (SNP) served as a positive control. ( D ) The intensity of target bands on the films was measured with the ImageJ software ( http://rsbweb.nih.gov/ij/ ). Semi-quantitative results are presented as mean ± SD and Student’s t-test was used for data analysis (Caspase-8, p = 0.875; Caspase-3, p = 0.686; Caspase-6, p = 0.486; Caspase-1, p = 0.343).

Article Snippet: Rabbit anti-caspase-1 (Santa Cruz, CA), anti-caspase-3 (Abcam, Cambridge, UK), anti-caspase-6, anti-caspase-8, mouse anti-IkBα (all from Cell Signaling Technology, MA) and anti-HMGB1 (Abcam) were used as primary antibodies.

Techniques: SDS Page, Western Blot, Positive Control, Software