mouse anti-src2  (Becton Dickinson)

Journal: BMC cancer

Article Title: STAMP alters the growth of transformed and ovarian cancer cells.

doi: 10.1186/1471-2407-10-128

Figure Lengend Snippet: Figure 4 Effect of elevated STAMP on the properties of GR induction of GREtkLUC reporter gene in 293 cells. (A) Dose-response curves for GR induction of GREtkLUC reporter with varying amounts of transiently transfected GR plasmid. S13 (containing stably transfected STAMP) and vector control (VA) cells were cotransfected with GREtkLUC reporter and the indicated amounts of GR-encoding plasmid. Dose-response curves were conducted and plotted as described by Tao et al. [32]. (B) Modulation of Amax, partial agonist activity of the antiglucocorticoid Dex- Mes (%DM), and EC50 by elevated STAMP. Four independent experiments with clone S13 and VA cells such as in A, which included assaying the activity of 1 μM Dex-Mes, were analyzed to yield Amax and EC50 in addition to partial agonist activity of 1 μM Dex-Mes (%DM). The average values ± S.E.M. are plotted. * P < 0.05, ** P < 0.005, *** P < 0.0005 for clone S13 vs. the similarly treated clone VA sample. (C) GR mRNA levels in VA and S13 cells. qRT-PCR was used as described in Materials and Methods to determine the level of GR mRNA in VA and S13 cells with or without 30 ng of transiently transfected GR plasmid (± S.E.M. of triplicates). (D) Western blot with TIF2 antibody of cell lysates from cells transfected with no, or 30 ng of GR plasmid. The lower panel shows the equal levels of the loading control, a-tubulin, in each sample.

Article Snippet: Mouse monoclonal anti-TIF2 antibody is from Bethyl Labs (#A300345A).

Techniques: Transfection, Plasmid Preparation, Stable Transfection, Control, Activity Assay, Quantitative RT-PCR, Western Blot

Journal:

Article Title: GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

doi: 10.1242/jcs.009878

Figure Lengend Snippet: Agarose-bound GST fusion proteins of ERRγ (1ug) were used to pull down whole cell lysates containing a fixed amount of SRC1 (A) or SRC2 (B), mixed with increasing amounts of the wild-type GNL3L (A1, B1) or the dI mutant lacking the ERRγ-interacting domain (A2, B2). Whole cell proteins in each sample were adjusted to the same amount. In the agarose-retained portions (R), the interaction between GNL3L and ERRγ can reduce the amount of SRC1 and SRC2 bound by ERRγ in a dose-dependent manner, but the dI mutant fails to do so. Conversely, when GST-ERRγ fusion proteins were used to pull down the same amount of GNL3L in the presence of increasing amounts of SRC1 (C) or SRC2 (D), SRC1 and SRC2 were able to reduce the amount of GNL3L bound by ERRγ in a dose-dependent way as well. Proteins in the agarose-bound fraction and in the supernatant are indicated by (R) and (S), respectively.

Article Snippet: Western analyses were performed using the mouse anti-Myc, rabbit anti-HA, rabbit anti-SRC1 (abcam, ab2859, 500X), and mouse anti-SRC2 (BD Transduction Laboratories, clone 29, 250X) antibodies.

Techniques: Mutagenesis

Journal:

Article Title: GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

doi: 10.1242/jcs.009878

Figure Lengend Snippet: (A) The GNL3L effect on the DNA binding of ERRγ was examined by electrophoretic mobility shift assays (EMSA) using ERE-containing probes and whole cell lysates expressing the indicated recombinant proteins. Compared to the probe alone (lane 1) and the vector-transfected control sample (lane 2), the ERRγ-specific DNA-protein complex can be identified in lane 3 (arrow b), competed by excess non-labeled probes (lane 4), and supershifted by anti-Myc antibody (lane 5, arrow a). Coexpression of GNL3L produces fast-moving complexes (lane 6, arrows d and e), which can be supershifted by anti-Myc antibody (lane 7, arrow c) but not by anti-HA antibody (lane 8). GNL3L itself cannot bind the ERE probe (lane 9). The intensity of the fast-moving complex d is reduced by a deletion of the ERRγ-binding I-domain of GNL3L (lanes 10–12). (B) The fast-moving complex d and the slow-moving complex b were retrieved from the EMSA gel, fractionated in SDS-denaturing PAGE, and analyzed for their ERRγ (α-Myc), GNL3L (α-HA), SRC1, and SRC2 protein components by western blottings. Our results indicate that the increase in the electrophoretic mobility of the ERRγ-DNA complex by GNL3L coexpression can be explained by a loss of SRC1 binding (arrow) and diminished SRC2 binding, rather than by protein cleavage of ERRγ.

Article Snippet: Western analyses were performed using the mouse anti-Myc, rabbit anti-HA, rabbit anti-SRC1 (abcam, ab2859, 500X), and mouse anti-SRC2 (BD Transduction Laboratories, clone 29, 250X) antibodies.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Expressing, Recombinant, Plasmid Preparation, Transfection, Labeling, Western Blot

Journal:

Article Title: GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

doi: 10.1242/jcs.009878

Figure Lengend Snippet: (A) Using the same cell-based reporter system as described in Fig. 4, we show that the ERE-specific transcriptional activity in cells coexpressing ERRγ and SRC1 (8.0±0.3) is 1.7 times higher than that of the ERRγ-expressing sample (4.8±0.3). When coexpressed with the wild-type GNL3L (WT), this ERRγ and SRC1-mediated ERE-specific transcriptional activity is reduced by 55 and 70 percent compared to the sample expressing both ERRγ and SRC1 in a dose-dependent manner. This inhibitory effect of GNL3L on the SRC1-mediated coactivation of ERRγ requires the I-domain of GNL3L, as a deletion of this domain (dI) fails to suppress the transcriptional activity of ERRγ and SRC1 (P value = 0.17). (B) Using the same approach, we show that GNL3L can also suppress the coactivator function of SRC2 on the ERRγ-dependent transcriptional activity in a dose-dependent (54% reduction for 100ng of GNL3L and 71% reduction for 200ng of GNL3L) and I-domain-dependent (P value = 0.58) manner. Error bars represent stand error of mean (s.e.m.). ***, P value < 0.0001.

Article Snippet: Western analyses were performed using the mouse anti-Myc, rabbit anti-HA, rabbit anti-SRC1 (abcam, ab2859, 500X), and mouse anti-SRC2 (BD Transduction Laboratories, clone 29, 250X) antibodies.

Techniques: Activity Assay, Expressing

Journal:

Article Title: GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

doi: 10.1242/jcs.009878

Figure Lengend Snippet: Our data reveal a novel mechanism that regulates the activity of ERR family genes by a nucleolar GTP-binding protein GNL3L. GNL3L decreases the transcriptional activity of ERR proteins. This event takes place in the nucleoplasm and does not require the nucleolar localization of GNL3L. The interaction between GNL3L and ERRγ displaces coactivators such as SRC1 and SRC2 from the ERRγ complex. The SRC-depleted ERRγ protein binds DNA without GNL3L, resulting in transcriptional inhibition. In this model, the nucleolar accumulation of GNL3L does not appear to affect its ability to suppress the transcriptional function of ERR proteins (grey arrows). Abbreviations for protein domains of GNL3L and ERR are explained in Fig. 2A and 2D.

Article Snippet: Western analyses were performed using the mouse anti-Myc, rabbit anti-HA, rabbit anti-SRC1 (abcam, ab2859, 500X), and mouse anti-SRC2 (BD Transduction Laboratories, clone 29, 250X) antibodies.

Techniques: Activity Assay, Binding Assay, Inhibition