Journal:

Article Title: Comprehensive Mutational Analysis of a Herpesvirus Gene in the Viral Genome Context Reveals a Region Essential for Virus Replication †

doi: 10.1128/JVI.78.15.8026-8035.2004

Figure Lengend Snippet: Functional analysis of M50 mutants. (A) Coprecipitation of IgM53 and M50/p35 deletion mutants. pCR3-IgM53 and M50-del1, M50-del2, M50-del3, M50-del4, and wt M50 were cotransfected into 293 cells. Cells were radioactively labeled, and protein complexes with IgM53 were precipitated with protein A-Sepharose. Samples were analyzed by SDS-PAGE. (B) Test for functionality of the M50 deletion mutants. For functional analysis, M50-del1, M50-del2, M50-del3, and M50-del4 were reinserted into pΔM50-GFP. BAC DNA was isolated and transfected into M2-10B4 cells. Ten days postinfection, cells were screened for plaque formation. The results are shown in the row below the depicted gel. (C) Coprecipitation of IgM53 and M50/p35 mutants affecting the potential binding motif. pCR3-IgM53 plus M50E56A, M50Y57A, M50Y53A, M50Y53,57A, or wt M50 was cotransfected into 293 cells. Cells were radioactively labeled, and protein complexes were precipitated with protein A-Sepharose. Samples were analyzed by SDS-PAGE. (D) Western blot analysis after pull-down of the M50-M53 complex. pCR3-IgM53 plus wt M50 M50Y53,57A, M50Y53A, M50E56A, or M50Y57A was cotransfected into 293 cells. Samples were analyzed by SDS-PAGE and plotted on membranes. M50/p35 was detected with an M50-specific antiserum in untreated cell lysate (L) and after pull-down of the M50/IgM53 complex with protein A-Sepharose (P). M50/p35-specific bands are indicated.

Article Snippet: NIH 3T3 fibroblasts (ATCC CRL 1658), M2-10B4 bone marrow stroma cells (ATCC CRL 1972), human 293 cells (ATCC CRL 1573), and mouse embryonic fibroblasts were propagated as described previously ( 23 , 26 ).

Techniques: Functional Assay, Labeling, SDS Page, Isolation, Transfection, Binding Assay, Western Blot