Journal:

Article Title: Cross Talk between Retinoic Acid Signaling and Transcription Factor GATA-2

doi: 10.1128/MCB.24.15.6824-6836.2004

Figure Lengend Snippet: Coimmunoprecipitation analysis of GATA-2-RARα interaction. (A) 293T cells were transfected with expression plasmids encoding the indicated proteins and treated with 1 μM RA [RA(+)] or diluent alone [RA(−)] 24 h after transfection. Cell lysates were prepared 24 h later, immunoprecipitated (IP) with anti-Flag antibody, and analyzed by Western blotting with anti-RARα (top) or anti-Flag (bottom) antibodies. Input (10%) nuclear extracts were analyzed as controls for the level of protein expression. Note that under these conditions GATA-2 binds RARα, irrespective of RA treatment. (B) Lysates of 293T cells transfected with the indicated expression plasmids were immunoprecipitated with anti-Flag antibody and analyzed by anti-GATA-2 (top) or anti-Flag (bottom) antibodies. (C) Cell lysates of 293T cells transfected with the expression plasmids for the indicated proteins were immunoprecipitated with anti-Flag antibody as described above. The precipitated proteins were analyzed by Western blotting with the indicated antibodies. Note that RXRα only weakly binds GATA-2. (D) Nuclear extracts of human myeloid KG1 cells were immunoprecipitated with anti-GATA-2 antibody. The precipitated materials were then analyzed by Western blotting with antibodies against GATA-2, RARα, and RXRα. Mouse IgG was used as a control. Input (10%) materials were used as controls. Molecular size markers are indicated on the right.

Article Snippet: Agarose-conjugated anti-GATA-2 antibodies, agarose-conjugated mouse Ig, and polyclonal antibodies against GATA-2, RARα, and RXRα were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot

Journal:

Article Title: Cross Talk between Retinoic Acid Signaling and Transcription Factor GATA-2

doi: 10.1128/MCB.24.15.6824-6836.2004

Figure Lengend Snippet: RARα can be recruited to GATA motifs in DNA through interaction with GATA-2. 293T cells were transfected with the expression plasmids encoding RARα (A) or RXRα (B) and Flag-tagged GATA-2 as indicated. Nuclear extracts of the cells were then prepared and incubated with biotinylated oligonucleotides harboring GATA motifs (wild-type [wt] GATA oligonucleotides) or biotinylated mutant oligonucleotides in which GATA motifs were changed to TTTA (mutant [mt] GATA oligonucleotides). The oligonucleotides were then recovered by streptavidin-agarose beads, and the copurified proteins were analyzed by Western blotting with anti-RARα or anti-RXRα (top) and anti-Flag (bottom) antibodies. Input (10% input) was used as a control. (C) Nuclear extracts of human myeloid KG-1 cells were incubated with biotinylated oligonucleotides harboring GATA motifs and pulled down with streptavidin-agarose beads. The pulled-down materials were analyzed by Western blotting with antibodies against GATA-2, RARα, and RXRα.

Article Snippet: Agarose-conjugated anti-GATA-2 antibodies, agarose-conjugated mouse Ig, and polyclonal antibodies against GATA-2, RARα, and RXRα were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Transfection, Expressing, Incubation, Mutagenesis, Western Blot

Journal:

Article Title: Cross Talk between Retinoic Acid Signaling and Transcription Factor GATA-2

doi: 10.1128/MCB.24.15.6824-6836.2004

Figure Lengend Snippet: RXRα inhibits recruitment of RARα to GATA-2-GATA motif DNA complex. 293T cells were transfected with an expression plasmid for either Flag-tagged GATA-2, RARα, or RXRα, separately. The resultant nuclear extracts were then mixed as indicated. Nuclear extract of the cells transfected with an empty vector was used to make the total amounts of nuclear proteins equal. The nuclear extracts containing the indicated proteins were then incubated with biotinylated oligonucleotides harboring GATA motifs (wild-type [wt] GATA), and the nucleotides were captured by streptavidin-agarose beads. The resultant copurified proteins were analyzed by Western blotting with anti-Flag, anti-RARα, and anti-RXRα antibodies. Nuclear extracts prior to mixing were analyzed as controls for appropriate expression of the proteins used (input; 10%). Biotinylated oligonucleotides in which GATA core recognition motifs were mutated to TTTA (mutant [mt] GATA) were used as controls.

Article Snippet: Agarose-conjugated anti-GATA-2 antibodies, agarose-conjugated mouse Ig, and polyclonal antibodies against GATA-2, RARα, and RXRα were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Western Blot, Mutagenesis

Journal:

Article Title: Cross Talk between Retinoic Acid Signaling and Transcription Factor GATA-2

doi: 10.1128/MCB.24.15.6824-6836.2004

Figure Lengend Snippet: RARα renders GATA-2-dependent reporter activity RA responsive. 293T cells were transfected with a luciferase reporter plasmid containing a double GATA motif in the mouse GATA-1 promoter (designated GATA-1/Luc.; 0.5 μg) (A and C) or the reporter containing two copies of double GATA sites in the mouse CD34 promoter (designated CD34x2/Luc.; 0.5 μg) (B), together with expression plasmids for GATA-2 (GATA-2/pMT2, 100 ng) and RARα (0.5 μg), a C-terminally truncated form of RARα (RARα 403; 0.5 μg), RXRα (0.5 μg), or an empty vector (0.5 μg). Cells were then treated with RA (1 μM; solid bar) or diluent alone (open bar) 24 h after transfection, and the luciferase activities were measured another 24 h later. Luciferase activities are standardized against Renilla luciferase activity from cotransfected control reporter (pRL-CMV-Renilla luciferase) and expressed as fold increases over the activity of the reporter alone. The mutant reporters in which core recognition sites were changed from GATA to TTTA (mutant GATA-1/Luc. and mutant CD34x2/Luc.) were used as controls. Data are shown as means ± standard deviations of triplicate samples. (D) Expression of the endogenous GATA1 locus was measured by semiquantitative RT-PCR in HEL cells after 24 h of incubation with RA (10−6 M) and compared with that in untreated control samples. Analysis of GATA-2 expression is also shown, and parallel analysis of β2-microglobulin provided a control for normalization of RNA levels. NTC; no-template control. (E) Expression of endogenous CD34 and GATA-1 was measured in FDCPmix A4 cells by semiquantitative RT-PCR after 48 h of incubation with Epo (1 U/ml), RA (10−6 M), Epo plus RA, or the RARα antagonist Ro 41-5253 (10−5 M) and compared with that in an untreated control sample. Analysis of GATA-2 expression is also shown, and parallel analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) provided a control for normalization of RNA levels. ND, not done.

Article Snippet: Agarose-conjugated anti-GATA-2 antibodies, agarose-conjugated mouse Ig, and polyclonal antibodies against GATA-2, RARα, and RXRα were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Incubation

Journal:

Article Title: Cross Talk between Retinoic Acid Signaling and Transcription Factor GATA-2

doi: 10.1128/MCB.24.15.6824-6836.2004

Figure Lengend Snippet: GATA-2 is not included in RARα-RXRα-DNA complexes. 293T cells were transfected with an expression plasmid for either RARα, RXRα, or Flag-tagged GATA-2, separately, and the resultant nuclear extracts were mixed as indicated. Nuclear extract of the cells transfected with the empty vector was used to make the total amount of nuclear extracts the same. The mixtures of the nuclear extracts were then incubated with biotinylated oligonucleotides harboring RARE of either the DR5 (wild-type [wt] DR5; lanes 2 and 6) or the DR2 (wt DR2; lane 4) type, and the nucleotides were captured with streptavidin-agarose beads. The copurified proteins were then analyzed by Western blotting with anti-RARα, anti-RXRα, and anti-Flag antibodies. Nuclear extracts prior to mixing were analyzed as controls for appropriate expression of the proteins used (10% input; lane 1). Biotinylated oligonucleotides in which core recognition sites of the RARα-RXRα complex were mutated from GGTTCA and AGTTCA to GGTAGT and AGTAGT, respectively, were used as controls (mutant [mt] DR5 and DR2, lanes 3 and 5). Note that in no combination was GATA-2 copurified with RARα-RXRα-RARE complexes. The amounts of RARα and RXRα bound to DR5 are similar whether or not GATA-2 is included (lanes 2 and 6).

Article Snippet: Agarose-conjugated anti-GATA-2 antibodies, agarose-conjugated mouse Ig, and polyclonal antibodies against GATA-2, RARα, and RXRα were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Western Blot, Mutagenesis