Journal:

Article Title: An ATF/CRE Element Mediates both EBNA2-Dependent and EBNA2-Independent Activation of the Epstein-Barr Virus LMP1 Gene Promoter

doi:

Figure Lengend Snippet: EBNA2 does not affect the level or phosphorylation state of c-Jun or ATF-2 in DG75 cells. The EBNA2 expression vector pEΔA6 or equivalent amounts of the pSV2gpt control plasmid or the E1A 13S expression vector were transfected together with the CD2 expression vector pE300CY6 in DG75 cells. The transfected cells were selected for their CD2 expression with magnetic beads. The cells were lysed and equal amounts of protein extract were analyzed by SDS-PAGE and immunoblotting. The antibodies used in panel A were anti-c-Jun, anti-phospho-c-Jun(Ser63), and anti-phospho-c-Jun(Ser73), and those used in panel B were anti-ATF-2 and anti-phospho-ATF-2(Thr71). NIH 3T3 cell extracts containing nonphosphorylated or phosphorylated forms of c-Jun (panel A, lanes 1 and 2) and ATF-2 (panel B, lanes 1 and 2), respectively, were used as controls of antibody activity. The anti-c-Jun(Ser73) antibody also detected JunD phosphorylated at the Ser100 residue.

Article Snippet: The membranes were incubated with rabbit anti-c-Jun antibodies (sc-822X), mouse anti-ATF-2 antibodies (sc-187X), or a human serum containing anti-EBNA2 antibodies in phosphate-buffered saline (PBS; 180 mM NaCl, 3.6 mM KCl, 11 mM Na 2 HPO 4 , 2.0 mM KH 2 PO 4 ) containing 0.5% nonfat dry milk and, after repeated washings in PBS, incubated with horseradish peroxidase-conjugated donkey anti-rabbit (Amersham Life Science), sheep anti-mouse (Amersham Life Science), or goat anti-human (Bio-Rad) antibodies.

Techniques: Expressing, Plasmid Preparation, Transfection, Magnetic Beads, SDS Page, Western Blot, Activity Assay

Journal:

Article Title: An ATF/CRE Element Mediates both EBNA2-Dependent and EBNA2-Independent Activation of the Epstein-Barr Virus LMP1 Gene Promoter

doi:

Figure Lengend Snippet: EBNA2 coimmunoprecipitates with the c-Jun/ATF-2 heterodimer. The EBNA2 expression vector pc(BYRF) or equivalent amounts of the pc control plasmid were transfected together with pc(c-Jun) and pc(ATF-2) and the CD2 expression vector pE300CY6 in DG75 cells. The transfected cells were selected for CD2 expression with magnetic beads. After lysis of the cells, the proteins were immunoprecipitated with specific antibodies and adsorption to protein A/G agarose, eluted, and analyzed by SDS-PAGE and immunoblotting. Cell extracts that had not been subjected to immunoprecipitation were analyzed in lanes 1 to 3, and control immunoprecipitations without the specific antibody but including the adsorption step with protein A/G were analyzed in lanes 10 to 12. Lanes: 1, DG75 cells transfected with pc(BYRF); 2, DG75 cells transfected with the pc plasmid; 3, B95-8 cells; 4, anti-ATF-2 precipitate from DG75 cells transfected with pc(BYRF); 5, anti-ATF-2 precipitate from DG75 cells transfected with the pc plasmid; 6, anti-ATF-2 precipitate from B95-8 cells; 7, anti-c-Jun precipitate from DG75 cells transfected with pc(BYRF); 8, anti-c-Jun precipitate from DG75 cells transfected with the pc plasmid; 9, anti-c-Jun precipitate from B95-8 cells; 10, protein A/G agarose eluate from DG75 cells transfected with pc(BYRF); 11, protein A/G agarose eluate from DG75 cells transfected with the pc plasmid; 12, protein A/G agarose eluate from B95-8 cells. Antibodies used for visualizing the proteins on the immunoblots were anti-ATF-2 (A), anti-c-Jun (B), and a human serum containing anti-EBNA2 antibodies (C). The positions of ATF-2, c-Jun, EBNA2, and immunoglobulin heavy chains (Ig H) are indicated by the solid arrowheads.

Article Snippet: The membranes were incubated with rabbit anti-c-Jun antibodies (sc-822X), mouse anti-ATF-2 antibodies (sc-187X), or a human serum containing anti-EBNA2 antibodies in phosphate-buffered saline (PBS; 180 mM NaCl, 3.6 mM KCl, 11 mM Na 2 HPO 4 , 2.0 mM KH 2 PO 4 ) containing 0.5% nonfat dry milk and, after repeated washings in PBS, incubated with horseradish peroxidase-conjugated donkey anti-rabbit (Amersham Life Science), sheep anti-mouse (Amersham Life Science), or goat anti-human (Bio-Rad) antibodies.

Techniques: Expressing, Plasmid Preparation, Transfection, Magnetic Beads, Lysis, Immunoprecipitation, Adsorption, SDS Page, Western Blot

Journal:

Article Title: An ATF/CRE Element Mediates both EBNA2-Dependent and EBNA2-Independent Activation of the Epstein-Barr Virus LMP1 Gene Promoter

doi:

Figure Lengend Snippet: Identification of the transcription factors interacting with the Sp and ATF/CRE motifs in LRS. (A) Nuclear extract of DG75 cells was incubated under binding conditions with a 32P-labelled double-stranded oligonucleotide corresponding to the −50 to −19 LRS region in the presence of a 300-fold molar excess of the competitor LRS −50/−19 with a mutated Sp site. Antibody supershifts were carried out by incubation with a goat polyclonal antibody against Sp1, a rabbit polyclonal antibody against Sp3, and a mixture of the antibodies, as indicated below the autoradiogram. The reaction mixtures were analyzed by EMSA. Three specific complexes are indicated by solid arrows, one designated Sp1 and two designated Sp3. Two bands that were not abolished by competition are indicated by the dotted arrows. The position of the anti-Sp1 antibody-shifted complex is shown by the solid arrowhead. (B and C) EMSA and antibody supershift analysis were performed by incubating nuclear extract of DG75 cells under binding conditions with a 32P-labelled double-stranded oligonucleotide corresponding to the LRS −50 to −19 region with a mutated Sp site and with antibodies as indicated below the autoradiogram. Two bands that were not abolished by competition are indicated by the dotted arrows. (B) Three specific complexes are indicated by solid arrows, two of which are designated ATF-1, CREB-1 since they contain both factors. The positions of the antibody complexes are indicated by an open arrowhead for the anti-CREB-1 shift and solid arrowheads for the anti-ATF-1 shifts. (C) The third of the three specific complexes indicated by solid arrows is identified as ATF-2, c-Jun. The positions of the immunologically shifted complexes are shown by the solid arrowheads for the anti-ATF-1 shifts and the open arrowhead for the anti-c-Jun shift.

Article Snippet: The membranes were incubated with rabbit anti-c-Jun antibodies (sc-822X), mouse anti-ATF-2 antibodies (sc-187X), or a human serum containing anti-EBNA2 antibodies in phosphate-buffered saline (PBS; 180 mM NaCl, 3.6 mM KCl, 11 mM Na 2 HPO 4 , 2.0 mM KH 2 PO 4 ) containing 0.5% nonfat dry milk and, after repeated washings in PBS, incubated with horseradish peroxidase-conjugated donkey anti-rabbit (Amersham Life Science), sheep anti-mouse (Amersham Life Science), or goat anti-human (Bio-Rad) antibodies.

Techniques: Incubation, Binding Assay