Journal: PLoS ONE
Article Title: Human Metapneumovirus Glycoprotein G Disrupts Mitochondrial Signaling in Airway Epithelial Cells
Figure Lengend Snippet: G protein blocks the interaction between RIG-I and MAVS. ( A ) 293 cells were transfected with plasmids encoding Flag-tagged RIG-I-N and V5-tagged G or their control vectors. Total cell lysates were immunoprecipitated with anti-V5 antibody followed by Western blot using anti-Flag antibody to detect RIG-I-N. Reverse immunoprecipitation was also done, where RIG-I-N was immunoprecipitated using anti-Flag antibody and G protein was then detected using anti-V5 antibody. Total cell lysates were subjected to Western blot to determine levels of hMPV G and RIG-I-N expression. ( B ) 293 cells were transfected with a fixed amount of plasmids encoding Flag-tagged RIG-I and EGFP-tagged MAVS, or their control vectors, and increasing concentrations of a plasmid expressing V5-tagged G. Total cell lysates were immunoprecipitated with anti-Flag antibody to pull down RIG-I, followed by Western blot using anti-EGFP or anti-V5 antibody to detect associated MAVS and G, respectively. Data are representative of two independent experiments.
Article Snippet: Cells were harvested 30 h post-transfection and immunoprecipitation was carried out using immunoprecipitation Kit from Roche (Cat # 11719, Indianapolis, IN) and an antibody against either V5 or Flag or an isotype antibody control, as previously described , .
Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation