Journal: International Journal of Molecular Sciences
Article Title: Bax Inhibitor-1 Acts as an Anti-Influenza Factor by Inhibiting ROS Mediated Cell Death and Augmenting Heme-Oxygenase 1 Expression in Influenza Virus Infected Cells
Figure Lengend Snippet: Overexpression and endogenous or exogenous expression of BI-1 or BI-1 ∆C in Madin-Darby Canine Kidney (MDCK) cells. ( A ) Scheme of the lentiviral constructs for the expression of wild type BI-1 or nonfunctional mutant BI-1 ΔC in MDCK cells. BI-1 or BI-1 ΔC was inserted into the lentiviral pEF vector. The pEF lentivirus containing wild type BI-1 or the non-functional mutant BI-1 ΔC was produced and used to infect MDCK cells. ( B ) Illustration of specific primer sets which was used to analyze endogenous or exogenous expression of BI-1 . ( C ) The endogenous or exogenous expression of BI-1 was confirmed by RT-PCR analysis in indicated cells; and the expression of HA was confirmed by western blot analysis in indicated cells. The expression of GAPDH and Actin was used as loading control. ( D ) The illustration of whole experimental protocol used in this study. (Abbreviations: chimeric Rous sarcoma virus (RSV) sequence, Rev response elements (RRE), central polypurine tract (cPPT), elongation factor 1 alpha (EF-1α) promoter region, a copGFP (copepod GFP) tag, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), cells growth media (CGM), virus growth media (VGM), and hemagglutinin assay (HA assay)).
Article Snippet: Cells, Virus, Reagents, and Lentiviral Infection Madin Darby Canine Kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC CCL-3, Manassas, VA, USA) and maintained in a minimum essential medium (MEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), and 100 U/mL penicillin/streptomycin (Gibco).
Techniques: Over Expression, Expressing, Construct, Mutagenesis, Plasmid Preparation, Functional Assay, Produced, Reverse Transcription Polymerase Chain Reaction, Western Blot, Sequencing, Hemagglutination Assay