Journal: Journal of Virology
Article Title: Calcium-Dependent Calpain Proteases Are Implicated in Processing of the Hepatitis C Virus NS5A Protein
Figure Lengend Snippet: Subcellular localization of the NS5A protein in transiently transfected cells. (A) Schematic representation of the entire NS5A protein and its major structural components. Representation of the amino acid composition of the NS5A fragments used in the subcellular localization studies. Plasmid pHPI 1406 encodes the NS5A protein that lacks the first 162 amino acids from the N terminus. Abbreviations: AD I, acidic domain I; ADII, acidic domain II; PRR, proline-rich region. Asp 154 ). (B) Vero or HuH-7 cells, seeded in 10-mm cover glasses, were transfected with either plasmid pHPI 728 (a, b, c, g, h, and i), pHPI 1406 (e and f), or with the empty plasmid pCI (d) and either left untreated (a, d, e, f, and g) or treated with MG132 (5 μΜ; b and h) or ALLN (10 μΜ; c and i), for the last 12 h. Cells were fixed at 36 h p.t. with 3.7% paraformaldehyde, and immunofluorescence analysis was performed by using the affinity-purified NS5A polyclonal antibody. Samples were examined by laser scanning confocal microscopy.
Article Snippet: Vero, WRL68, and HuH-7 cells were maintained in Dulbecco's modified Eagle medium (Biochrom KG) supplemented with 5 (Vero) or 10% (WRL68 and HuH-7) fetal bovine serum (Gibco BRL), penicillin and streptomycin (at concentrations of 5 IU ml−1 and 50 mg ml−1 , respectively), 2 mM l -glutamine, and nonessential amino acids (only for HuH-7) (1×; Biochrom KG).
Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Affinity Purification, Confocal Microscopy