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Results: 7

1.
Figure 1

Figure 1. Western blot detection of recombinant GST-lamin A processing by purified caspases.. From: A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C.

(A) The indicated concentration of caspase-6 was incubated with GST-lamin A for two hours. (B) 300 Units of caspases 1–9 were incubated with GST-lamin A for two hours. Intact and cleaved lamin A were detected via western blotting using anti-GST antibody.

Robert Mintzer, et al. PLoS One. 2012;7(1):e30376.
2.
Figure 3

Figure 3. Effect of staurosporine on the generation of cleaved lamin A/C in SKNAS cells.. From: A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C.

SKNAS cells were treated with the indicated concentration of staurosporine for 6 hours prior to detection of the small lamin A/C cleavage product as described in Experimental Procedures. The assay was performed in triplicate one time. The mean and standard error of the mean are reported.

Robert Mintzer, et al. PLoS One. 2012;7(1):e30376.
3.
Figure 4

Figure 4. Apoptosis-mediated cleavage of lamin A/C is elevated in wild-type relative to caspase-6 KO fibroblasts.. From: A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C.

Fibroblasts derived from caspase-6 KO (▪) or wild type (•) mice were treated with the indicated concentration of staurosporine for 6 hours prior to detection of the small lamin A/C cleavage product. The assay was performed in quadruplicate two times with similar results; mean and standard error of the mean are reported.

Robert Mintzer, et al. PLoS One. 2012;7(1):e30376.
4.
Figure 2

Figure 2. Western blot detection of lamin A/C from SKNAS neuroblastoma cells upon staurosporine treatment.. From: A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C.

(A) Schematic of the N- and C-terminal globular domains of Lamin A/C with VEID-containing central α-helical region as the site of caspase-6 proteolysis. (B) SKNAS cells were treated with DMSO control or staurosporine for 6 hours prior to cell lysis. Lysates were probed for small lamin A/C subunit (Lanes 1–2), large lamin A/C subunit (Lanes 3–4) or total lamin A/C (Lanes 5–6). (C) SKNAS cells were treated with DMSO or staurosporine for the indicated time prior to cell lysis. Lysates were probed for large lamin A/C subunit (red) or β-Actin (green).

Robert Mintzer, et al. PLoS One. 2012;7(1):e30376.
5.
Figure 5

Figure 5. Effect of staurosporine on the release of Lactate Dehydrogenase in SKNAS cells.. From: A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C.

SKNAS cells were treated with the indicated concentration of staurosporine for 2 (•), 4 (▪), 6 (▴) or 8 (♦) hours prior to detection of LDH release to the cell supernatant. The assay was performed in triplicate and represents 1 of at least 2 experiments with similar results. The data was normalized to fold increase over DMSO treatment. The mean and standard error of the mean are reported.

Robert Mintzer, et al. PLoS One. 2012;7(1):e30376.
6.
Figure 7

Figure 7. Effect of peptide-based caspase inhibitors on SKNAS cells as determined using the Caspase-Glo® 6 assay.. From: A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C.

SKNAS cells were treated with Ac-VEID-CHO (•) or Ac-DEVD-CHO (▪) prior to addition of 3 µM staurosporine for 6 hours and detection of VEID-ase activity as described in Experimental Procedures. Concentration inhibition curves were performed in duplicate and represent 1 of at least 3 experiments with similar results. Concentration-response curves for each inhibitor were normalized to zero and 100% based on no staurosporine or DMSO, respectively. The mean and standard error of the mean are reported.

Robert Mintzer, et al. PLoS One. 2012;7(1):e30376.
7.
Figure 6

Figure 6. Effect of peptide-based caspase inhibitors on the generation of cleaved lamin A/C in SKNAS cells.. From: A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C.

(A) SKNAS cells were treated with z-VEID-FMK (♦), z-DEVD-FMK (▪), Q-VD-OPh (•), Ac-VEID-CHO (▴) or Ac-DEVD-CHO (○) prior to addition of 3 µM staurosporine for 6 hours. (B) SKNAS cells were treated with z-ID-TFPM (•), z-EID-TFPM (▴) or z-VEID-TFPM (▪) prior to addition of 3 µM staurosporine for 6 hours. Detection of the small lamin A/C cleavage product was performed as described in Experimental Procedures. Concentration inhibition curves were performed in duplicate and represent 1 of at least 3 experiments with similar results. Concentration-response curves for each inhibitor were normalized to zero and 100% based on no staurosporine or DMSO, respectively. The mean and standard error of the mean are reported.

Robert Mintzer, et al. PLoS One. 2012;7(1):e30376.

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