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1.

Figure. From: Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family.

Fig. 8. The octapeptide motif of Pax5 mediates repression by Grg4. Transient transfection experiments with the expression plasmids indicated were performed in SP2/0 cells and subsequently evaluated as described in the legend to Figure .

Dirk Eberhard, et al. EMBO J. 2000 May 15;19(10):2292-2303.
2.

Figure. From: Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family.

Fig. 4. In vitro binding of Pax5 to Grg4. (A) C-terminal deletions of GST–Grg4 proteins. (B) The SP domain of Grg4 interacts with Pax5. GST pull-down assays were used to study the interaction between in vitro translated, 35S-labeled Pax5 protein and GST (lane 2) or GST–Grg4 proteins (lanes 3–7) bound to glutathione–Sepharose. Lane 1 contained 10% of the Pax5 protein input. (C) The octapeptide of Pax5 mediates binding of Grg4. The 35S-labeled Pax proteins indicated were analyzed for binding to GST or GST–Grg4. The input lane contained ∼10% of the total Pax protein used in each assay.

Dirk Eberhard, et al. EMBO J. 2000 May 15;19(10):2292-2303.
3.

Figure. From: Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family.

Fig. 10. Model of Grg4 recruitment by Pax5 (BSAP). (A) Summary of the identified interactions between Pax5 and Grg4. (B) Model ofGrg-mediated gene repression by Pax5. As different members of the Grg family are co-expressed with Pax5 throughout midbrain and B-cell development (; D.Eberhard, unpublished data), we hypothesize that Pax5 can stably recruit Grg proteins to a specific promoter only in collaboration with a second Grg-binding transcription factor (X). CoA, coactivator protein(s); BTM, basal transcription machinery.

Dirk Eberhard, et al. EMBO J. 2000 May 15;19(10):2292-2303.
4.

Figure. From: Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family.

Fig. 5. In vivo interaction of Groucho and Pax5. Myc-tagged Grg4 or Groucho proteins were expressed in transiently transfected COP-8 fibroblasts either alone (lanes 1, 3 and 5) or with a Flag-tagged Pax5 protein (lanes 2, 4 and 6). After 48 h, whole-cell lysates were prepared and immunoprecipitated with the anti-Flag M2 antibody. Immuno complexes were analyzed by Western blotting first with a polyclonal anti-Myc antibody and then with a polyclonal antiserum directed against the Pax5 paired domain (). Lanes 1–4 contained ∼7.5% of the total input (I) protein or supernatant (S), respectively. P, precipitated protein.

Dirk Eberhard, et al. EMBO J. 2000 May 15;19(10):2292-2303.
5.

Figure. From: Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family.

Fig. 2. Schematic diagram of mutant Pax5 proteins. (A) Domain structure of Pax5 and extent of amino acid deletion in the different mutant proteins. (B) The octapeptide sequence is shown together with the Y179E mutation and the ΔOP and Δ9A deletions. (C) Conservation of the octapeptide motif. The octapeptide sequences of mouse (m), zebrafish (zf) and Drosophila (d) members of the Pax2/5/8 family are aligned with the corresponding Engrailed homology region 1 (eh1) of the homeodomain transcription factors En2 and Goosecoid (Gsc). Identical amino acids are highlighted by black overlay. For sequences, see , and .

Dirk Eberhard, et al. EMBO J. 2000 May 15;19(10):2292-2303.
6.

Figure. From: Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family.

Fig. 6. Induced phosphorylation of Grg4 upon interaction with Pax5. (A) Pax5-dependent phosphorylation of Grg4. Pax5 (where indicated) and Myc-tagged Grg4 were transiently expressed in COP-8 cells for 48 h. Grg4 was then precipitated from whole-cell lysates with a monoclonal anti-Myc antibody, incubated with λ protein phosphatase (λ-Pase) and then analyzed by Western blotting with a polyclonalanti-Myc antibody. (B) Specificity of the Grg4 phosphorylation. Myc-tagged Grg4 and Flag-tagged Pax5 or En2 proteins were co-expressed in COP-8 cells followed by Western blot analysis with anti-Myc and anti-Flag antibodies, respectively. The asterisk denotes a cross-reacting protein. (C) The phosphorylation of Grg4 depends on direct interaction with Pax2/5/8 proteins. The Pax proteins indicated were co-expressed with Myc-tagged Grg4 in COP-8 cells followed by Western blotting with polyclonal anti-Myc and anti-paired domain antibodies. (D) Phosphorylation of Myc-tagged Grg4 in NIH 3T3 and COS-7 cells co-expressing Pax5. (E) Endogenous (endo) Grg proteins are phosphorylated in Pax5-expressing COP-8 cells, as shown by Western blot analysis with a pan-TLE (Grg) antibody ().

Dirk Eberhard, et al. EMBO J. 2000 May 15;19(10):2292-2303.
7.

Figure. From: Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family.

Fig. 3. The N-terminal region of Grg4 interacts with the transactivation domain of Pax5. (A) Interaction of Pax5 and VP16-Grg4. The expression plasmids indicated (100 ng) were co-transfected into J558L cells together with the Renilla luciferase gene pRL-SV40 (0.4 µg) and firefly luciferase gene luc-CD19 (5 µg; ). After 48 h, the cells were lysed, luciferase activities were measured, and the activity of the firefly luciferase was standardized relative to the control Renilla luciferase to normalize for differences in transfection efficiencies. Luciferase values are shown relative to the activity measured with the empty expression vector pKW2T (left bar). (B) Interaction of the Q domain of Grg4 with the transactivation domain of Pax5. Both luciferase genes and the Pax5 expression plasmids indicated (100 ng each) were electroporated into J558L cells with (hatched bars) or without (black bars) the VP16-Grg4 expression vector (100 ng). Average values of three experiments are shown.

Dirk Eberhard, et al. EMBO J. 2000 May 15;19(10):2292-2303.
8.

Figure. From: Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family.

Fig. 1. Interaction of Pax5 with the N-terminal region of Grg4 in yeast. (A) Schematic diagram of Pax5, Grg4 and the respective fusion proteins. The different domains of each protein are indicated together with the corresponding amino acid positions. The chimeric Gal4–Pax5 protein was used as a bait to isolate the VP16-Grg4 fusion protein in a yeast two-hybrid screen of a cDNA expression library that was generated by fusing the transactivation domain (TAD) of VP16 to cDNA derived from 9.5/10.5-day-old mouse embryos (). See text for description of the Grg4 structure. PD, paired domain; OP, octapeptide; HD, partial homeodomain; TA, trans activation region; ID, inhibitory domain; DBD, DNA-binding domain. (B) Specific interaction of Pax5 and Grg4 in yeast. Expression plasmids of the combinations indicated (central panel) were transformed into yeast and selected for by growth on plates lacking leucine and tryptophan (left panel). Activation of a Gal4-dependent HIS3 gene was examined by growth on selection plates additionally lacking histidine (right panel).

Dirk Eberhard, et al. EMBO J. 2000 May 15;19(10):2292-2303.
9.

Figure. From: Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family.

Fig. 9. The octapeptide motif functions as a repression domain in Drosophila embryos. (A) Schematic diagram of the Hairy derivatives used in the Sxl repression assay. The HairyPax2.1 protein was generated by fusing the Hairy sequences at amino acid 268 to a 90-amino-acid sequence (143–232) of the zebrafish Pax2.1 protein encompassing the conserved octapeptide motif (). The HairyGsc protein containing the eh1/GEH motif of Goosecoid (Gsc) was described previously (). (BE) Sxl repression by chimeric Hairy proteins in female blastoderm embryos of Drosophila. The HairyGsc (C) and HairyPax2.1 (D and E) proteins were expressed under the control of the hunchback (hb) promoter in wild-type (wt, C and D) or groE48mat– mutant embryos (E). Sxl expression was analyzed in transgenic (C–E) and control (B) female embryos by whole-mount staining with a monoclonal antibody detecting the female-specific form of Sxl (). (F) Effects of HairyGsc and HairyPax2.1 expression on female viability. Wild-type females were crossed with hb transgenic males, and the number of viable progeny is indicated.

Dirk Eberhard, et al. EMBO J. 2000 May 15;19(10):2292-2303.
10.

Figure. From: Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family.

Fig. 7. Grg4 represses the transcriptional activity of Pax5. The Pax expression vector (0.5 µg), luciferase genes luc-CD19 (5 µg) and pRL-SV40 (0.4 µg), and increasing amounts of the indicated Grg expression vector were used for transient transfection of SP2/0 cells (A–D and G–I). The amount of expression plasmid was equalized by the addition of pKW2T, and all data were evaluated as described in the legend to Figure A. Normalized luciferase values of one representative experiment (A and B) or three independent transfections (C–I) are shown relative to the luciferase activity measured in the absence of the Pax protein. (A) Repression of the Pax5 transactivation function by Grg4. (B) Failure of Grg4 to repress the function of a paired domain (PD)-VP16 protein. (C) Inability of the Grg4-ΔWD40 protein to repress the transcriptional activity of Pax5. (D) Grg5 fails to antagonize Grg4-mediated repression of Pax5 activity. (E) Repression of a chromatinized reporter gene by Grg4. Expression vectors (1 µg) encoding Gal4–Pax5 or the Gal4 DNA-binding domain (DBD) were transfected together with a Grg4 expression plasmid (0.5 µg) into U2-OS cells containing an integrated luciferase gene, followed by luciferase analysis as described (). (F) Grg4-mediated repression of the rat TPO promoter. HeLa cells were transfected with mPax8a (0.1 µg) and Grg4 (0.5 µg) expression plasmids and the luciferase gene TPO-luc (2.5 µg) and pRL-SV40 (10 ng). ΔTPO-luc contains a mutated Pax8-binding site (). (G and H) Grg4-mediated repression of the tran scriptional activity of zebrafish zfPax2.1 and Drosophila dPax258. (I) Repression of the Pax5 transactivation function by Drosophila Groucho.

Dirk Eberhard, et al. EMBO J. 2000 May 15;19(10):2292-2303.

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