view release on metacpan or  search on metacpan

t/data/demos/15037632.cp1252  view on Meta::CPAN

F IG. 2. Interaction of FOG-1 zinc fingers with TACC3. a, zinc fingers 2-4 of FOG-1 interact with the coiled-coil domain of TACC3 in vitro. GST alone or GST-TACC3cc-bound glutathione-Sepharose beads were incubated with in vitro expressed 35 S-labeled...



GST Pull-down Assay —Recombinant pGBKT7 FOG-1 zf 2-4 (aa 299-388) was transcribed using T7 RNA polymerase (Roche Applied Science) and translated using wheat germ extract (Promega) according to the manufacturer's instructions. Bacteria (BL21) (Stratag...



Cell Culture and Benzidine Assay —Cell lines were cultured in 5% CO 2 at 37 °C. MEL cells and 293T cells were cultured in Dulbecco's modified Eagle's low glucose medium ( Invitrogen ) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 100 u...



Immunoprecipitation Assay —For immunoprecipitation assay in MEL cells, whole cell extracts were prepared from actively proliferating MEL HA-FOG-1 cells (which stably express HA-tagged FOG-1 protein under the control of an EF1 promoter) and control ME...



Protein Localization Assays —293T cells were plated onto 10-cm plates. Each plate was transfected with 5 µg of Qiagen column-purified plasmid and 15 µl of FuGENE 6 transfection reagent (Roche Applied Science). Cells were harvested 48 h post-transfect...



DNA Transfections —Qiagen column-purified DNA was transfected into cells using FuGENE 6 transfection reagent at a 3:1 DNA:FuGENE 6 ratio according to manufacturer's instructions. Cells were plated onto 10-mm wells to a density of 50%. For reporter as...



Retroviral Infection and Sorting —Vesicular stomatitis virus G protein-pseudotyped retroviral particles were generated via transient transfection of the stable packaging cell line 293 GPG ( 30 ) of the MMP-based retroviral vector. MEL or G1ER cells (...



Reverse Transcription-PCR —RNA was extracted from cell lines using TriReagent (Sigma) and reverse-transcribed using oligo(dT) and Moloney murine leukemia virus reverse transcriptase (Promega). Oligonucleotides used to amplify the -major globin ( 31 )...



RESULTS

TACC3 Interacts with the Zinc Fingers 2-4 of FOG-1 —To identify potential FOG-1-interacting proteins, we used an N-terminal region of FOG-1 (including fingers 1-4) as bait in a yeast two-hybrid screen. Four independent clones, repeated 32, 1, 2, and ...







View larger version (43K):

[in this window]

[in a new window]

F IG. 1. Isolation of TACC3 as a FOG-1-interacting protein in a yeast two-hybrid assay. A MEL cDNA library was screened using as bait the zinc fingers 1-4 of FOG-1 (aa 250-388) and the yeast strain AH109. Candidate clones were re-transformed into AH1...



In Vitro and in Vivo Association of FOG-1 and TACC3 —To examine further the specificity of the interaction between FOG-1 and TACC3, in vitro interactions were assessed by GST pull-down experiments. As shown in Fig. 2 a , in vitro expressed [ 35 S]met...



Interaction of endogenous FOG-1 and TACC3 was also examined in mammalian cells. For this purpose, a stable cell line derived from MEL cells that express an HA-tagged form of FOG-1 (or HA alone for control cells) to a level similar to that seen for th...



FOG-1 Changes Its Intracellular Localization in the Presence of TACC3 —FOG-1 and GATA-1 interact in the nucleus to control gene expression. However, TACC3 localizes in the cytoplasm and perinuclear region in interphase cells and associates with the c...







View larger version (31K):

[in this window]

[in a new window]

F IG. 3. TACC3 sequesters FOG-1 in the cytoplasm. 293T cells were transfected with GATA-1 ( lane 1 ), GATA-1/FOG-1 ( lane 2 ), GATA-1/FOG-1/TACC3 ( lane 3 ), FOG-1/TACC3 ( lane 4 ), and GATA-1/TACC3 ( lane 5 ) and harvested 48 h post-transfection, an...



In summary, these experiments indicate that expression of TACC3 induces the targeted clearance of FOG-1 from the nucleus but does not affect the localization of GATA-1 or the unrelated transcription factor P/CAF. Furthermore, co-expression of GATA-1 ...



GATA-1 / FOG-1 and FOG-1 / TACC3 Interactions Are Competitive —To test the effect of GATA-1 on the association between FOG-1 and TACC3 as well as the effect of TACC3 on the interaction between GATA-1 and FOG-1, co-immunoprecipitation experiments were...







View larger version (45K):

[in this window]

[in a new window]

F IG. 4. GATA-1 and TACC3 compete for their interaction with FOG-1. 293T cells were transiently transfected with a total of 5 µg of DNA consisting of different combinations of expression plasmids for GATA-1, HA-FOG-1, and TACC3 as indicated in the fi...



TACC3 Affects GATA-1-mediated Transcription —Interaction between GATA-1 and FOG-1 is essential for the normal gene expression profile and differentiation of erythroid and megakaryocytic cell lineages ( 7, 10 ). FOG-1 acts either as a co-activator or ...







View larger version (31K):

[in this window]

[in a new window]

F IG. 5. TACC3 blocks FOG-1 function as a transcriptional co-repressor for GATA-1. a, 293T cells were transfected with a total of 200 ng of DNA with final amount of DNA maintained constant by the addition of empty expression vector when required. Equ...



To ascertain whether the observed effects are due to direct interaction between FOG-1 and TACC3 and not to a nonspecific effect of TACC3 on transcription, we expressed increasing amounts of the FOG-1 zinc finger cluster (zinc fingers 2-4) in the repo...