stat3 mammalian expression vector  (OriGene)

Journal: Scientific Reports

Article Title: EGF-mediated reduced miR-92a-1-5p controls HTR-8/SVneo cell invasion through activation of MAPK8 and FAS which in turn increase MMP-2/-9 expression

doi: 10.1038/s41598-020-68966-4

Figure Lengend Snippet: Effect of STAT1 and STAT3 silencing and inhibition by Fludarabine and Stattic in HTR-8/SVneo cells on the expression of miR-92a-1-5p in presence or absence of EGF. ( a ) Expression of miR-92a-1-5p by qRT-PCR in HTR-8/SVneo cells transfected with STAT1 siRNA treated with or without EGF for 24 h as compared to untreated scrambled control siRNA transfected cells. ( b ) Expression of miR-92a-1-5p by qRT-PCR in HTR-8/SVneo cells transfected with STAT3 siRNA treated with or without EGF for 24 h as compared to untreated scrambled control siRNA transfected cells. ( c ) Expression of miR-92a-1-5p by qRT-PCR in HTR-8/SVneo cells pre-treated with or without Fludarabine (4 h) and subsequently treated with or without EGF for 24 h. ( d ) Expression of miR-92a-1-5p by qRT-PCR in HTR-8/SVneo cells pre-treated with or without Stattic (1 h) and subsequently treated with or without EGF for 24 h. Each bar represents relative expression after normalization with the miR-191 used as an internal miRNA normalizer. Data in ( a ), ( b ), ( c ) and ( d ) is mean ± SEM of three independent experiments performed in triplicates. Synthetically synthesized STAT3 binding sites present in the promoter region of miR-92a-1-5p (BS1 corresponding to 492–510 nt and BS2 corresponding to 777–795 nt position from miRNA precursor encoding region, table and ) were cloned upstream of the PKG promoter of the firefly luciferase gene in pmirGLO Dual-Luciferase vector. ( e ) shows the relative luciferase activity determined from HEK-293T cells co-transfected with clones possessing the synthetically synthesized STAT3 binding sites in pmirGLO Dual-Luciferase vector with STAT3 mammalian expression vector, as indicated. The data are represented as the mean of three experiments ± S.E.M. performed in duplicates.

Article Snippet: Alternately, HEK-293T cells were co-transfected with 250 ng of luciferase reporter plasmid harboring the STAT3 binding site along with 310 ng of STAT3 mammalian expression vector (OriGene Technologies, MD, USA) using Lipofectamine RNAiMAX reagent in OptiMEM.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Transfection, Synthesized, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay

Journal: Scientific Reports

Article Title: EGF-mediated reduced miR-92a-1-5p controls HTR-8/SVneo cell invasion through activation of MAPK8 and FAS which in turn increase MMP-2/-9 expression

doi: 10.1038/s41598-020-68966-4

Figure Lengend Snippet: Graphical representation of the effect of reduced miR-92a-1-5p expression during EGF-mediated increased HTR-8/SVneo cell invasion. EGF leads to reduced expression of miR-92a-1-5p in HTR-8/SVneo trophoblastic cells; reduced miR-92a-1-5p leads to increased invasion of HTR-8/SVneo cells by targeting MAPK8 and FAS, which increases expression of MMP-2 & MMP-9, and decreases expression of TIMP1. EGF activated STAT1 and STAT3 may lead to reduced expression of miR-92a-1-5p by binding to the miRNA precursor encoding region as inhibition of STAT1 and STAT3 abrogates reduction of miR-92a-1-5p expression in EGF treated HTR-8/SVneo cells.

Article Snippet: Alternately, HEK-293T cells were co-transfected with 250 ng of luciferase reporter plasmid harboring the STAT3 binding site along with 310 ng of STAT3 mammalian expression vector (OriGene Technologies, MD, USA) using Lipofectamine RNAiMAX reagent in OptiMEM.

Techniques: Expressing, Binding Assay, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

doi: 10.1084/jem.20110283

Figure Lengend Snippet: STAT3 somatic mutations identified in inflammatory hepatocellular tumors

Article Snippet: A full-length STAT3 open reading frame cloned in pCMV6-XL4 vector was purchased from OriGene (SC124165; available from GenBank/EMBL/DDBJ under accession no. NM_139276 ).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

doi: 10.1084/jem.20110283

Figure Lengend Snippet: Gain-of-function mutations in STAT3 in IHCA. (A) Distribution of STAT3 mutations identified in IHCA is represented according to the different protein domains. Asterisk indicates the two mutations that are found on the same allele in tumor #379. Stat3-C location is indicated in gray. (B) STAT3 mutants L78R (L 78 ), E166Q (E 166 ), Y640F (Y 640 ), D502Y K658Y (D 502 /K 658 ), G656_Y657insF (G 656 ), Y657_M660dup (Y 657 ), and Stat3-C (S C ) or control STAT3 WT (WT) and empty plasmid (EP) were transfected in Hep3B, HepG2, and Huh7 cells expressing a STAT3-driven luciferase (Luc) reporter construct. STAT3 activation was measured after 6 h of serum starvation and, when indicated, was treated for 3 h with 100 ng/ml IL-6. Shown is the Luc activity (mean) determined from triplicate co-transfections (± SD) relative to pSIEM-luc alone (EP) without IL-6. (C) Graphs are qRT–PCR results showing the induction of endogenous CRP , STAT3 , and SOCS3 mRNA after overexpression of mutant STAT3 relative to unstimulated EP-transfected Hep3B cells (EP; mean ± SD). (D) Endogenous CRP mRNA expression in Hep3B cells transfected with increasing amounts of expression plasmids encoding WT or E 166 STAT3 mutant (dotted line: mock-transfected cells). (E) Activity of the STAT3 mutants, including D502Y (D 502 ), K658Y (K 658 ), and the double mutant D502Y K658Y (D 502 /K 658 ) were evaluated using STAT3-driven Luc in Hep3B cell line. Data are from triplicate transfections (± SD) relative to pSIEM-luc alone (EP). *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student’s t test.

Article Snippet: A full-length STAT3 open reading frame cloned in pCMV6-XL4 vector was purchased from OriGene (SC124165; available from GenBank/EMBL/DDBJ under accession no. NM_139276 ).

Techniques: Plasmid Preparation, Transfection, Expressing, Luciferase, Construct, Activation Assay, Activity Assay, Quantitative RT-PCR, Over Expression, Mutagenesis, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

doi: 10.1084/jem.20110283

Figure Lengend Snippet: Subcellular localization and role of tyrosine 705 phosphorylation in STAT3 IHCA mutants. (A) Immunochemistry of phospho-STAT3-Y705 in STAT3-mutated IHCA and adjacent nontumor liver (NTL). (B–D) Vectors driving the expression of STAT3 mutants, including L78R (L 78 ), E166Q (E 166 ), Y640F (Y 640 ), G656_Y657insF (G 656 ), or WT (WT) STAT3, were transfected into Hep3B cells. (B) Expression of total and phosphorylated ( p Y705) STAT3 proteins were assessed by Western blot and bands were quantified with ChemiDoc using the Quantity One software; bars show the expression of p Y705 STAT3 relative to total STAT3. (C and D) Subcellular localization of total and phosphorylated ( p Y705) STAT3 proteins were analyzed by Western blot (C) and by immunofluorescence (D). Results are representative of three independent experiments. Bars, 20 µm. (E) CRP mRNA expression after transfection of WT, Y705F (Y 705 ), Y640F (Y 640 ), Y640F/Y705F (Y 640 /Y 705 ), G656_Y657insF (G 656 ), or G656_Y657insF/Y705F (G 656 /Y 705 ) STAT3 in unstimulated Hep3B cells. Data are from triplicate transfections (± SD) relative to EP. *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student’s t test.

Article Snippet: A full-length STAT3 open reading frame cloned in pCMV6-XL4 vector was purchased from OriGene (SC124165; available from GenBank/EMBL/DDBJ under accession no. NM_139276 ).

Techniques: Expressing, Transfection, Western Blot, Software, Immunofluorescence, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

doi: 10.1084/jem.20110283

Figure Lengend Snippet: Dimerization of STAT3 IHCA mutants. (A) Flag- and Myc-tagged constructs encoding either WT or mutant Y640 STAT3 were co-transfected (1:1) into Hep3B cells treated, or not treated, with 100 ng/mlIL-6. STAT3 dimer formation was detected after immunoprecipitation using the anti-Flag antibody, followed by immunoblot (IB) analysis with Myc, Flag, and p Y705-STAT3–specific antibodies. Results are representative of three independent experiments. (B) Effects of increasing WT STAT3 expression (wedge) on the constitutive activity of Y 640 mutant STAT3 without IL-6. The graph plots the mean level of expression of SOCS3 and CRP relative to cells transfected with EP (± SD). Units are plasmid micrograms. (C) Several STAT3 IHCA mutations cluster near the TAD of STAT3 (crystal structure; NCBI Protein database accession no. 1BG1; ). Residues 640 and 656–660 (shown in green), which are mutated in IHCA tumors, the phosphorylated tyrosine (Tyr-705, black), and TAD residues 711–716 (blue), are shown in stick representation. One polypeptide chain and both TADs are shown as a cartoon. The twofold related molecule, except the TAD, is shown as a surface representation. For residues of the TAD, Tyr-710 of the twofold related molecule is labeled, whereas residues 711–716 are not, for clarity. (D) DNA-binding activity of 10 µg of nuclear extracts of Hep3B cells engineered to express WT, Y 640 , or G 656 STAT3 mutants. Bars indicate the DNA-binding activity to consensus STAT3 oligonucleotides relative to activity measured in cells transfected with EP. *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student’s t test. Data are from triplicate transfections (±SD).

Article Snippet: A full-length STAT3 open reading frame cloned in pCMV6-XL4 vector was purchased from OriGene (SC124165; available from GenBank/EMBL/DDBJ under accession no. NM_139276 ).

Techniques: Construct, Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Expressing, Activity Assay, Plasmid Preparation, Labeling, Binding Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

doi: 10.1084/jem.20110283

Figure Lengend Snippet: Role of IL-6–JAK pathway and Src kinases on STAT3 IHCA mutant activation. STAT3 mutants Y 640 and E 166 were overexpressed in Hep3B cells. Activation of STAT3 is shown by Luc activity (mean) determined from triplicate transfections (± SD) relative to pSIEM-luc alone (EP) with serum starvation. (A) Hep3B cells were exposed to increasing concentrations of IL-6. (B) STAT3 mutants (E 166 and Y 640 ) or empty vector (EP) were co-transfected with JAK1 siRNA (+) or control siRNA (−) into Hep3B cells exposed to 100 ng/ml IL-6. (C) STAT3 mutants (E 166 and Y 640 ) or empty vector (EP) were co-transfected with gp130 siRNA (+) or control siRNA (−) into Hep3B cells. (D) STAT3 mutants (E 166 and Y 640 ) or empty vector (EP) were co-transfected with JAK2 siRNA (+) or control siRNA (−) into Hep3B cells. (E) STAT3 mutants (E 166 and Y 640 ) or empty vector (EP) were co-transfected with JAK1 siRNA (+) or control siRNA (v) into Hep3B cells. (F) STAT3 mutants (E 166 and Y 640 ) or empty vector (EP) were transfected into Hep3B cells exposed to Src inhibitor 1 (SrcI-1; 10 µM; 9 h). *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student’s t test. Data are from triplicate transfections (± SD). (G) Expression profiles of STAT3-mutated IHCA and gp130-mutated IHCA. Comparison was performed for the 28 genes validated by qRT-PCR that were significantly differentially expressed between IHCA and nontumorous livers. 5 STAT3-mutated IHCAs (black) were compared with 11 gp130-mutated IHCAs (in white). Results are expressed as mean ± SD; differences between groups are not significant (two-tailed Mann-Whitney test).

Article Snippet: A full-length STAT3 open reading frame cloned in pCMV6-XL4 vector was purchased from OriGene (SC124165; available from GenBank/EMBL/DDBJ under accession no. NM_139276 ).

Techniques: Mutagenesis, Activation Assay, Activity Assay, Transfection, Plasmid Preparation, Two Tailed Test, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal:

Article Title: S -Glutathionylation Impairs Signal Transducer and Activator of Transcription 3 Activation and Signaling

doi: 10.1210/en.2008-1241

Figure Lengend Snippet: Reversible reduction in IL-6-induced STAT3 signaling in response to mild oxidative stress. A, HepG2 cells were serum starved for 3–4 h and then left untreated or treated with PDTC (100 μm, 2 h) or diamide (500 μm, 30 min), followed by stimulation with IL-6 (20 ng/ml) for 20 min at 37 C. B, Cells were pretreated with diamide (500 μm) for 30 min, washed, and incubated with fresh medium for 0, 5, 15, and 45 min before the addition of IL-6 for 15 min. A and B, Cells were lysed and analyzed by Western blot with an antibody raised against pY-STAT3 (top panels). ERK1/2 was used as a loading control (lower panels). The OD of the pY-STAT3 band in untreated, IL-6-stimulated cells was arbitrarily given the value of 1.0. The numbers given under the different panels [relative units (Rel. Units)] represent the relative intensities of the protein bands from two independent experiments. The positions of pY-STAT3 and ERK1/2 are indicated on the left and that of the molecular mass markers (in kDa) are shown on the right. C, PDTC decreases nuclear translocation of STAT3 in HepG2 cells. Cells were preincubated with vehicle (veh.) or 50 μm PDTC for 2 h, then treated with 20 ng/ml IL-6 for 20 min. Cells were fixed and probed with STAT3 antibody to detect subcellular localization of STAT3. Nuclei were visualized by staining with Topro. Confocal images in the bottom panels are overlay of STAT3 and Topro images. The results are representative to three independent experiments. D, Effect of PDTC on FBG protein levels of HepG2 cells stimulated with IL-6. HepG2 cells were left untreated or treated with PDTC for 2 h before the addition of IL-6 (20 ng/ml) or IL-1β (20 ng/ml) for 24 h. Cells were lysed and analyzed by immunoblotting for expression of FBG (top panel), pY-STAT3 (middle panel), and ERK1/2 (lower panel) as a loading control. Similar results were obtained in two independent experiments.

Article Snippet: Full-length cDNA encoding human STAT3 (pCMV6-XL4; OriGene Technologies, Inc., Rockville, MD) was subcloned in-frame into N-terminal p3xFLAG-CMV (Sigma-Aldrich Corp., St. Louis, MO) to generate the Flag-STAT3 fusion construct.

Techniques: Incubation, Western Blot, Translocation Assay, Staining, Expressing

Journal:

Article Title: S -Glutathionylation Impairs Signal Transducer and Activator of Transcription 3 Activation and Signaling

doi: 10.1210/en.2008-1241

Figure Lengend Snippet: STAT3 contains cysteine residues that are targets for oxidant-mediated S-glutathionylation. A and B, Serum-starved HepG2 cells were left untreated or incubated either with PDTC (50 μm, 2 h), GSH disulfide (GSSG, 20 mm, 30 min) or diamide (500 μm, 30 min), after which cells were homogeneized in RIPA buffer supplemented with the thiol biotinylating agent, MBB (200 μm), for 30 min on ice. A, After quenching the alkylation reaction with excess l-cysteine, the clarified cell lysates were subjected to immunoprecipitation with STAT3 antibody and immunoblotted with streptavidin-conjugated HRP (top panel) and STAT3 (lower panel) as a loading control. B, Alternatively, the clarified cell lysates were incubated with CaptAvidin-linked agarose for 1 h at 4 C, and the immobilized proteins were eluted with biotin, followed by Western blot analysis with anti-STAT3. The band intensity of thiol-biotinylated STAT3 in untreated cells was arbitrarily given the value of 1.0. Results are representative of two separate experiments with similar results. C, Reversible oxidation of STAT3 cysteine residues by PDTC. HepG2 cells were treated without or with PDTC for 2 h. Cell lysates were subjected to biotin switch assay, followed by affinity precipitation with streptavidin-agarose and then immunoblot analysis with anti-STAT3 antibody. Bands were detected only in PDTC-treated cells and when biotin-HPDP was included in the assay. D, HepG2 cells were left untreated or incubated with PDTC, GSSG, or diamide as indicated in A. STAT3 immunoprecipitates were separated by SDS-PAGE under nonreducing conditions, and analyzed by Western blot with a monoclonal antibody against protein-bound GSH (αSSG, top panel) and STAT3 (lower panel) as a loading control. The S-glutathionylated STAT3 band intensity in untreated cells was arbitrarily given the value of 1.0. Rel. Units, Relative units; veh, vehicle.

Article Snippet: Full-length cDNA encoding human STAT3 (pCMV6-XL4; OriGene Technologies, Inc., Rockville, MD) was subcloned in-frame into N-terminal p3xFLAG-CMV (Sigma-Aldrich Corp., St. Louis, MO) to generate the Flag-STAT3 fusion construct.

Techniques: Incubation, Immunoprecipitation, Western Blot, Biotin Switch Assay, Affinity Precipitation, SDS Page

Journal:

Article Title: S -Glutathionylation Impairs Signal Transducer and Activator of Transcription 3 Activation and Signaling

doi: 10.1210/en.2008-1241

Figure Lengend Snippet: In vitro S-glutathionylation inhibits JAK2-mediated STAT3 phosphorylation and its DNA-binding activity. A, STAT3 is an in vitro target of glutathionylation/deglutathionylation reaction. Anti-STAT3 immunoprecipitates from control HepG2 cells were left untreated (lane 1) or treated with diamide (1 mm)/2 mm GSH for 1 h at room temperature (lanes 2–5), after which vehicle (veh.), recombinant E. coli GRX-1 or DTT was added for 30 min. The immune pellets were resolved by SDS-PAGE under nonreducing conditions, and analyzed by Western blot with antibodies raised against αSSG (top panel) and STAT3 as a loading control (bottom panel). B, STAT3 immunoprecipitates were left untreated or treated with 1 mm diamide/GSH as described under Materials and Methods. Tyrosine phosphorylation was then performed using recombinant active JAK2 for 10 min at 30 C. The proteins in the immune pellets were analyzed by Western blot using antibodies against pY-STAT3 (upper panel) and total STAT3 (lower panel). The reversibility of the inhibitory effect of diamide was shown by using 2 mm DTT before the JAK2-mediated phosphorylation step. The numbers given under the panel [relative units (Rel. Units)] represent the quantitated ratios of pY-STAT3 to total STAT3 relative to that of untreated vehicle control. Results are representative of two independent experiments. C, Cells were treated in the absence or the presence of IL-6 (20 ng/ml) for 30 min. Nuclear extracts were prepared, and DNA binding activity of STAT3 was determined as described under Materials and Methods. Nuclear extracts were incubated with agarose-bound oligonucleotide probe containing the wild-type (wt) GAS consensus sequence (lanes 1–2) or the same probe mutated (mut) at the GAS site (lanes 3–4). Bound STAT3 was resolved by SDS-PAGE, and analyzed by Western blot with anti-pY-STAT3. An identical result was obtained in an additional experiment. D, The nuclear extracts of IL-6-stimulated cells were treated without or with 3 mm GSSG for 1 h at 37 C to induce in vitro S-glutathionylation. STAT3 immobilized with agarose-bound wild-type oligonucleotide probe was analyzed by Western blot using anti-pY-STAT3. The numbers given in the panel (Rel. Units) represent the intensities of pY-STAT3 protein bands relative to that of control IL-6-stimulated cells. Results are representative of four independent observations from two separate experiments with similar results.

Article Snippet: Full-length cDNA encoding human STAT3 (pCMV6-XL4; OriGene Technologies, Inc., Rockville, MD) was subcloned in-frame into N-terminal p3xFLAG-CMV (Sigma-Aldrich Corp., St. Louis, MO) to generate the Flag-STAT3 fusion construct.

Techniques: In Vitro, Binding Assay, Activity Assay, Recombinant, SDS Page, Western Blot, Incubation, Sequencing

Journal:

Article Title: S -Glutathionylation Impairs Signal Transducer and Activator of Transcription 3 Activation and Signaling

doi: 10.1210/en.2008-1241

Figure Lengend Snippet: Ectopic expression of GRX-1 enhances IL-6-induced STAT3 signaling. Cells were transiently transfected with Flag-STAT3 together with pcDNA vector or GRX-1 plasmid. A, Forty-eight hours later, cells were homogeneized in RIPA buffer containing MBB, and anti-Flag immunoprecipitates were analyzed by Western blot using HRP-conjugated streptavidin (Strep.) (top panel) or anti-STAT3 (second panel) and anti-GRX-1 (bottom panel) antibodies. B, Serum-starved transfected cells were left untreated or incubated with diamide (500 μm) for 30 min, followed by the addition of IL-6 (20 ng/ml) for 20 min. The anti-Flag immunoprecipitates were analyzed by Western blotting for the detection of pY-STAT3 (top panel), and Flag-STAT3 (second panel). Immunoblot analysis showed detection of GRX only in clarified lysates of GRX-1-transfected cells (third panel). Endogenous ERK 1/2 was detected at comparable levels in each lane (bottom panel). The pY-STAT3 band intensity in Flag-STAT3-transfected cells stimulated with IL-6 was arbitrarily given the value of 1.0. No pY-STAT3 signal was detected in the absence of IL-6 (data not shown). IP, Immunoprecipitation; IB, immunoblots. C, The levels of Flag-STAT3 in the nuclear (Nuc) (second panel) and cytosolic (Cyt) (third panel) fractions of serum-starved cells exposed to IL-6 (20 ng/ml) for 30 min were assessed by Western blot analysis. The membranes were reprobed with BRG-1 (top panel) and ERK 1/2 (bottom panel) antibodies to demonstrate equal protein loading in each lane. D, Nuclear Flag-STAT3 band intensities were normalized to cellular pool (nuclear + cytosolic) of Flag-STAT3 protein. The dot plot represents the individual measurements that were derived from two separate experiments, each performed in duplicate dishes. Rel. Units, Relative units; veh., vehicle.

Article Snippet: Full-length cDNA encoding human STAT3 (pCMV6-XL4; OriGene Technologies, Inc., Rockville, MD) was subcloned in-frame into N-terminal p3xFLAG-CMV (Sigma-Aldrich Corp., St. Louis, MO) to generate the Flag-STAT3 fusion construct.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Incubation, Immunoprecipitation, Derivative Assay

Journal:

Article Title: S -Glutathionylation Impairs Signal Transducer and Activator of Transcription 3 Activation and Signaling

doi: 10.1210/en.2008-1241

Figure Lengend Snippet: Effect of GRX-1 overexpression on STAT3 DNA binding and induction of target genes. A, ChIP assays using agarose-conjugated Flag antibody (M2-Ag) were performed in HepG2 cells transfected with Flag-STAT3 plasmid together with empty vector (pcDNA) or GRX-1 plasmid, and then left untreated or stimulated with IL-6 for 30 min. The results are expressed as percentage of immunoprecipitated (IP) DNA to total DNA input (input). Similar results were obtained in three independent experiments. B, HepG2 cells were transiently cotransfected with pGAS-TA-Luc reporter plasmid, pCMVSport-β-galactosidase expression plasmid, and Flag-tagged STAT3 together with either pcDNA empty vector or GRX-1 plasmid. Twenty-four hours later, the cells were serum starved for 4 h and then pretreated with vehicle (veh.) (open bars) or 20 ng/ml IL-6 (filled bars) for 6 h. Cell extracts were analyzed for luciferase activity and normalized for β-galactosidase. All values are expressed relative to that of Flag-STAT3-expressing cells without IL-6. Results are expressed as means ± sd of a single experiment performed in triplicate dishes. Results are representative of two separate experiments with similar results.

Article Snippet: Full-length cDNA encoding human STAT3 (pCMV6-XL4; OriGene Technologies, Inc., Rockville, MD) was subcloned in-frame into N-terminal p3xFLAG-CMV (Sigma-Aldrich Corp., St. Louis, MO) to generate the Flag-STAT3 fusion construct.

Techniques: Over Expression, Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Luciferase, Activity Assay