Journal: Journal of Virology
Article Title: The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis †
Figure Lengend Snippet: (A) Time course of M45 expression during MCMV replication as determined by immunoblotting. Whole-cell lysates of mock-infected and MCMV-infected NIH 3T3 cells at various times after infection were separated by SDS-PAGE, transferred to a membrane, and probed with the anti-M45 antiserum or with the anti-actin monoclonal antibody. Lanes: 1, mock-infected cells; 2, 6 hpi; 3, 9 hpi; 4, 12 hpi; 5, 18 hpi; 6, 24 hpi; 7, 36 hpi; 8, 48 hpi. (B) Effect of PFA on M45 expression. Whole-cell extracts were prepared at 18 hpi (lanes 1 and 2), 24 hpi (lanes 3 and 4), or 48 hpi (lanes 5 and 6) from MCMV-infected NIH 3T3 cells treated with PFA (250 μg/ml) after virus adsorption (lanes 2, 4, and 6) or left untreated (lanes 1, 3, and 5). Protein expression was analyzed by immunoblotting with the anti-M45 antiserum or with the anti-actin monoclonal antibody. (C) Subcellular localization of M45 in MCMV-infected NIH 3T3 cells at 48 hpi, detected by immunofluorescence and confocal microscopy. Cells were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. (D) Subcellular localization of transiently expressed M45 protein in NIH 3T3 cells transfected with the pcDNA3-45 vector at 24 h posttransfection. For immunofluorescence and confocal microscopy analysis, cells were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. The merged pictures are shown.
Article Snippet: For immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P membranes (Millipore).
Techniques: Expressing, Infection, SDS Page, Adsorption, Immunofluorescence, Confocal Microscopy, Incubation, Transfection, Plasmid Preparation