Journal: PLoS ONE
Article Title: HCV Induces Telomerase Reverse Transcriptase, Increases Its Catalytic Activity, and Promotes Caspase Degradation in Infected Human Hepatocytes
doi: 10.1371/journal.pone.0166853
Figure Lengend Snippet: (A) Left panel, log phase Huh 5.15NS replicons were incubated with the pancaspase inhibitor (z-vad-fmk) (200uM) and at various times cellular lysates were assayed on WB for TERT or NS3-4A using C-terminal specific antibody. In right panel, log phase Huh 5.15NS replicons were incubated with indicated amounts of the specific Caspase 7 inhibitor (Millipore 28832) for 48 hr. Cellular lysates were then assayed on WB for TERT using C-terminal specific antibody. In the lower panel, log phase Huh 5.15NS replicons were incubated with pancaspase inhibitor or vehicle control for the indicated times and relative telomerase activity was determined in the cellular lysates as compared to day 0 using Real-time TRAP-RT PCR. *[Pancaspase inhibitor > control days 2, 3, and 4, p< 0.01] (B) Huh-7.5 cells were either infected with HCVcc (right panel) or mock control (left panel) and on various days WB were performed to detect TERT, NS3, and un-activated full length caspases 6 and 7 as well as cleaved fragments as indicated. # Antibodies recognizing both full length and upper cleaved caspase fragments were from Cell Signaling (Caspase 7 #9492 and Caspase 6 #9762). (C) Huh-7.5 or Huh-7 cells were infected with HCVcc and on various days assayed for TERT as well as lower kD cleaved fragments of caspase 6 and 7 by WB. Cell Signaling *ASP 162 antibody (#9761) was used to detect cleaved caspase 6 and **ASP 198 antibody (#8438) was used to detect cleaved caspase 7 with products appearing at 18 kD in both cases.
Article Snippet: WB experiments showed that the amount of 45 kD fragment was markedly reduced while the 120 kD monomer was bolstered in replicon cultures incubated with either the pancaspase inhibitor, Z-vad-fmk, or the specific caspase 7 inhibitor, Millipore 28832, ( , left and right panels respectively).
Techniques: Incubation, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Infection
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![(A) Left panel, log phase Huh 5.15NS replicons were incubated with the pancaspase inhibitor (z-vad-fmk) (200uM) and at various times cellular lysates were assayed on WB for TERT or NS3-4A using C-terminal specific antibody. In right panel, log phase Huh 5.15NS replicons were incubated with indicated amounts of the specific <t>Caspase</t> <t>7</t> inhibitor (Millipore 28832) for 48 hr. Cellular lysates were then assayed on WB for TERT using C-terminal specific antibody. In the lower panel, log phase Huh 5.15NS replicons were incubated with pancaspase inhibitor or vehicle control for the indicated times and relative telomerase activity was determined in the cellular lysates as compared to day 0 using Real-time TRAP-RT PCR. *[Pancaspase inhibitor > control days 2, 3, and 4, p< 0.01] (B) Huh-7.5 cells were either infected with HCVcc (right panel) or mock control (left panel) and on various days WB were performed to detect TERT, NS3, and un-activated full length caspases 6 and 7 as well as cleaved fragments as indicated. # Antibodies recognizing both full length and upper cleaved caspase fragments were from Cell Signaling (Caspase 7 #9492 and Caspase 6 #9762). (C) Huh-7.5 or Huh-7 cells were infected with HCVcc and on various days assayed for TERT as well as lower kD cleaved fragments of caspase 6 and 7 by WB. Cell Signaling *ASP 162 antibody (#9761) was used to detect cleaved caspase 6 and **ASP 198 antibody (#8438) was used to detect cleaved caspase 7 with products appearing at 18 kD in both cases.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5869/pmc05215869/pmc05215869__pone.0166853.g006.jpg)