Journal: Frontiers in Cellular and Infection Microbiology
Article Title: Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP
Figure Lengend Snippet: CacyBP bound to C-terminal domains of V protein. (A) Identification of protein interaction partners of V protein by yeast two-hybrid screening of a CEF cDNA library. (B–D) Co-localization of CacyBP with V, VC, and VN proteins in DF-1 cells. DF-1 cells were plated on coverslips and transfected with Flag-V, Flag-VN and Flag-VC. Forty-eight hours after transfection, the cells were stained with mouse anti-FLAG and rabbit anti-CacyBP antibodies, which was followed by staining with donkey anti-rabbit Alexa Fluor® 488 (green) and goat anti-mouse Alexa Fluor ®; 594 (Red) as secondary antibodies. The nucleus was subsequently stained with Hoechst 33342, and the images were captured using an ANDOR Revolution WD confocal microscope. Pearson's coefficient were analysis by Imaris (Microscopy Image Analysis Software, Bitplane, Switzerland), and Pearson's coefficient > 0.5 were considered to be probable co-localization. (E–G) HEK-293T cells in 60 mm cell culture dishes were co-transfected with the pCAGEN-Flag-V (or Flag-VN or Flag-VC) and pCMV-HA-CacyBP expression plasmids. Transfected cells were harvested and lysed 48 h after transfection, and the experiment was conducted according to the manufacturer's instructions for the Co-Immunoprecipitation Kit. After washing, immunoprecipitated proteins were identified and analyzed by western blotting using anti-HA or anti-FLAG antibodies.
Article Snippet: The specific primary antibodies of rabbit anti-CacyBP/SIP (1:100, BOSTER, China), mouse anti-FLAG (1:500, Invitrogen, USA) or mouse anti-HA (1:500, Invitrogen, USA) were used as appropriate.
Techniques: Two Hybrid Screening, cDNA Library Assay, Transfection, Staining, Microscopy, Software, Cell Culture, Expressing, Immunoprecipitation, Western Blot