Induction of Inflammation by West Nile virus Capsid through the Caspase-9 Apoptotic Pathway
Joo-Sung Yang*1, Mathura P. Ramanathan*1, Karuppiah Muthumani*, Andrew Y. Choo*, Sung-Ha Jin*, Qian-Chun Yu*, Daniel S. Hwang*, Daniel K. Choo*, Mark D. Lee*, Kesen Dang*, J. Joseph Kim†, David B. Weiner* , and WaixingTang
Figure 5. Apoptosis determining domain in WNV-Cp gene. a, Schematic diagram of pcWNV-Cp-DJY, pcWNV-CpWT, and pcWNV-Cp∆3′ constructs. b, Immunoprecipitation of in vitro translated protein from pcWNV-Cp-DJY (Cp-DJY), pcWNV-CpWT (CpWT), and pcWNV-Cp-3′ (Cp∆3′) plasmids. As a negative control, pcDNA3.1 in vitro translated supernatants were analyzed. c, Colorimetric caspase-3 activity assay using pcWNV-Cp-DJY (Cp-DJY), pcWNV-CpWT (CpWT), or pcWNV-CpΔ3′ (CpΔ3’) plasmid–transfected cells. d, The cell lysates were assayed for caspase-9-like activity, and the pcDNA3.1-transfected cell lysate was used as the negative control. e, Inhibition of WNV-Cp–induced apoptosis by a dominant negative (DN) caspase-9 plasmid (DN Casp9) was assayed with equal amount of cell lysates from co-transfection of pcWNV-Cp-DJY (Cp-DJY) or pcWNV-CpWT (CpWT); an expression level of pro-caspase-9 cleavage products (35–37 kD) was compared to 3′-terminal deletion mutant, pcWNV-Cp∆3′ (Cp∆3′) and pcDNA3.1 by Western blot analysis with anti-human caspase-9 mAb.
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