Journal: Cells

Article Title: Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

doi: 10.3390/cells11203184

Figure Lengend Snippet: Cardiac-specific Dsg2 knockout induced cardiac fibrosis. ( A ) Masson staining of heart sections in WT and CS-Dsg2 −/− (−/−) mice. Arrow shows cardiac fibrosis. Collagen volume fraction in the hearts of WT and CS-Dsg2 −/− mice was assessed. ( B ) Representative Western blots from mouse left ventricular (LV), interventricular septum (IVS), and right ventricle (RV). DSG2, PPARα, pSTAT3, pSMAD3, pAKT, α-SMA, and Collagen I were detected using specific antibodies. STAT3, SMAD3, AKT, and GAPDH were used as loading controls. ( C ) Results of quantitative PCR analysis of PPARα, TGF-β, α-SMA, and Collagen I mRNA levels in mouse LV and RV are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT.

Article Snippet: For transient transfection, cells were plated at optimal densities and grown for 24 h. Cells were then transfected with Dsg2 siRNA (MBS828119, MyBioSource, San Diego, CA, USA) or Stat3 siRNA (6354, Cell Signaling Technology) using lipofectamine reagent according to the manufacturer’s instructions.

Techniques: Knock-Out, Staining, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Cells

Article Title: Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

doi: 10.3390/cells11203184

Figure Lengend Snippet: Effects of Dsg2 siRNA and Stat3 siRNA on the expression levels of fibrotic markers in HL-1 cells. ( A , B ) HL-1 cells were transfected with control siRNA or Dsg2 siRNA. ( A ) Representative Western blots for DSG2, pSTAT3, α-SMA, and Collagen I were detected using specific antibodies. STAT3 and β-actin were used as loading controls. ( B ) Results of quantitative PCR analysis of Dsg2, α-SMA, and Collagen I mRNA levels in HL-1 cells treated with control or Dsg2 siRNA are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 3. * p < 0.05, ** p < 0.01 vs. control. ( C , D ) HL-1 cells were transfected with control siRNA or Stat3 siRNA. ( C ) Representative Western blots for STAT3 and α-SMA were detected using specific antibodies. β-actin were used as loading controls. ( D ) Results of quantitative PCR analysis of α-SMA and Collagen I mRNA levels in HL-1 cells treated with control or Stat3 siRNA are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 3. * p < 0.05, ** p < 0.01, vs. control.

Article Snippet: For transient transfection, cells were plated at optimal densities and grown for 24 h. Cells were then transfected with Dsg2 siRNA (MBS828119, MyBioSource, San Diego, CA, USA) or Stat3 siRNA (6354, Cell Signaling Technology) using lipofectamine reagent according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

doi: 10.3390/cells11203184

Figure Lengend Snippet: Fenofibrate alleviated cardiac fibrosis in CS-Dsg2 −/− mice. ( A ) Masson staining of heart sections in WT, CS-Dsg2 −/− mice, and CS-Dsg2 −/− mice treated with fenofibrate (Dsg2 −/− F). Collagen volume fraction in the hearts of WT, CS-Dsg2 −/− , and Dsg2 −/− F mice were assessed. ( B ) Representative Western blots from ventricles of WT, CS-Dsg2 −/− , and Dsg2 −/− F mice. DSG2, PPARα, pSTAT3, pSMAD3, pAKT, α-SMA, and Collagen I were detected using specific antibodies. STAT3, SMAD3, AKT, and GAPDH were used as loading controls. ( C ) Results of quantitative PCR analysis of PPARα, TGF-β, α-SMA, and Collagen I mRNA levels in mouse ventricles are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Article Snippet: For transient transfection, cells were plated at optimal densities and grown for 24 h. Cells were then transfected with Dsg2 siRNA (MBS828119, MyBioSource, San Diego, CA, USA) or Stat3 siRNA (6354, Cell Signaling Technology) using lipofectamine reagent according to the manufacturer’s instructions.

Techniques: Staining, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Cells

Article Title: Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

doi: 10.3390/cells11203184

Figure Lengend Snippet: AAV9-Pparα alleviated cardiac fibrosis in CS-Dsg2 −/− mice. ( A ) Masson staining of heart sections in CS-Dsg2 −/− mice and CS-Dsg2 −/− mice received AAV9-Pparα (Dsg2 −/− P). Collagen volume fraction in the hearts of CS-Dsg2 −/− and Dsg2 −/− P mice were assessed. ( B ) Representative Western blots from ventricles of CS-Dsg2 −/− and Dsg2 −/− P mice. PPARα, pSTAT3, pSMAD3, pAKT, α-SMA, and Collagen I were detected using specific antibodies. STAT3, SMAD3, AKT, and GAPDH were used as loading controls. ( C ) Results of quantitative PCR analysis of PPARα, TGF-β, α-SMA, and Collagen I mRNA levels in mouse ventricles are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CS-Dsg2 −/− .

Article Snippet: For transient transfection, cells were plated at optimal densities and grown for 24 h. Cells were then transfected with Dsg2 siRNA (MBS828119, MyBioSource, San Diego, CA, USA) or Stat3 siRNA (6354, Cell Signaling Technology) using lipofectamine reagent according to the manufacturer’s instructions.

Techniques: Staining, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Communications biology

Article Title: PRMT5-mediated methylation of STAT3 is required for lung cancer stem cell maintenance and tumour growth.

doi: 10.1038/s42003-024-06290-7

Figure Lengend Snippet: Fig. 1 | PRMT5 expression levels are increased in lung cancer cells. a, b PRMT5 expression is elevated in a lung adenocarcinoma and b lung squamous cell carci- noma. RNA-seq data from The Cancer Genome Atlas (TCGA) were analysed on the UCSC Xena browser. c Correlation between STAT3 and PRMT5 protein levels in 12 NSCLC cell lines (NCI-H23, LU65, CORL105, CHAGOK1, NCI-H3255, NCI-

Article Snippet: STAT3 activation was monitored by western blotting using an anti-phosphorylated STAT3 (Y705) antibody (CST; 9138).

Techniques: Expressing, RNA Sequencing

Journal: International Journal of Molecular Medicine

Article Title: GFI1 promotes the proliferation and migration of esophageal squamous cell carcinoma cells through the inhibition of SOCS1 expression

doi: 10.3892/ijmm.2021.5017

Figure Lengend Snippet: GFI1 enhances NF-κB and STAT3 activity by inhibiting SOCS1 expression in esophageal squamous cell carcinoma cell lines. (A and B) siGFI1 was transfected into KYSE30 and KYSE150 cells. (A) mRNA and (B) protein expression levels of SOCS1 were examined using reverse transcription-quantitative PCR and western blotting, respectively. (C) NF-κB activity, (D) p-p65 and (E) p-STAT3 protein levels were analyzed in KYSE30/KYSE150 cells following transfection with siGFI1. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. si, small interfering RNA; GFI1, growth factor-independent 1; SOCS1, suppressor of cytokine signaling 1; p-, phosphorylated.

Article Snippet: Rabbit polyclonal antibodies against phosphorylated (p)-p65 (1:1,000; cat. no. 310012) were obtained from Chengdu Zen Bioscience Co., Ltd. Rabbit monoclonal antibodies against STAT3 (1:1,000; cat. no. 4904S) and p-STAT3 (1:1,000; cat. no. 4113S) were purchased from Cell Signaling Technology, Inc.

Techniques: Activity Assay, Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: GFI1 promotes the proliferation and migration of esophageal squamous cell carcinoma cells through the inhibition of SOCS1 expression

doi: 10.3892/ijmm.2021.5017

Figure Lengend Snippet: SOCS1 inhibits the increase in esophageal squamous cell carcinoma cell proliferation and migration induced by GFI1. (A-H) siSOCS1 was transfected into the GFI1-knockdown KYSE30 and KYSE150 cells. (A) Colony formation assays were performed on the double transfected cells. (B) Cell migration was determined using Transwell assays. (C) NF-κB activity, (D) p-p65, p-STAT3 and SOCS1 protein levels were analyzed. (E-G) Ratio of p-p65 to p65 and STAT3 to p-STAT3, as well as (H) the semi-quantification of SOCS1 protein expression were determined using ImageJ. Magnification, ×200. * P<0.05, ** P<0.01 *** P<0.001 vs. control; # P<0.05, ## P<0.01 and ### P<0.001 vs. siGFI1. SOCS1, suppressor of cytokine signaling 1; si, small interfering RNA; GFI1, growth factor-independent 1; p-, phosphorylated.

Article Snippet: Rabbit polyclonal antibodies against phosphorylated (p)-p65 (1:1,000; cat. no. 310012) were obtained from Chengdu Zen Bioscience Co., Ltd. Rabbit monoclonal antibodies against STAT3 (1:1,000; cat. no. 4904S) and p-STAT3 (1:1,000; cat. no. 4113S) were purchased from Cell Signaling Technology, Inc.

Techniques: Migration, Transfection, Knockdown, Activity Assay, Expressing, Control, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: GFI1 promotes the proliferation and migration of esophageal squamous cell carcinoma cells through the inhibition of SOCS1 expression

doi: 10.3892/ijmm.2021.5017

Figure Lengend Snippet: Regulatory model of the role of GFI1 in ESCC progression. The results of the present study demonstrated that the upregulation of GFI1 promoted ESCC proliferation and migration via inhibition of SOCS1 expression, leading to the activation of the NF-κB and STAT3 pathways. GFI1, growth factor-independent 1; ESCC, esophageal squamous cell carcinoma; SOCS1, suppressor of cytokine signaling 1.

Article Snippet: Rabbit polyclonal antibodies against phosphorylated (p)-p65 (1:1,000; cat. no. 310012) were obtained from Chengdu Zen Bioscience Co., Ltd. Rabbit monoclonal antibodies against STAT3 (1:1,000; cat. no. 4904S) and p-STAT3 (1:1,000; cat. no. 4113S) were purchased from Cell Signaling Technology, Inc.

Techniques: Migration, Inhibition, Expressing, Activation Assay

Journal: BMC Cancer

Article Title: Cucurbitacin-I (JSI-124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B Leukemic Cells

doi: 10.1186/1471-2407-11-268

Figure Lengend Snippet: JSI-124-induced apoptosis was not mediated by c-Jun activation in B-cell malignancies . A . BJAB, I-83, and NALM-6 cells were pre-treated with 20 μM SP600125 followed by 1 μM JSI-124. Decreased levels of phospho-and total c-Jun protein levels were detected by western blotting in the cells treated with the combination of SP600125 and JSI-124 compared to SP600125 alone. Representative original data from three independent experiments are shown. The XIAP and STAT3proteins level, as determined by immunoblotting with antibodies against XIAP and serine phosphor-STAT3/STAT3, did not change with this treatment B . Apoptotic cell population was measured using flow cytometric analysis for AnnexinV/PI staining for apoptosis after 24 hour treatment with JSI-124, either alone or in combination with SP600125. DMSO-treated cells were taken as the control. Cells that were 7-AAD-negative and Annexin V-positive were undergoing apoptosis. The percentage of cells in each quadrant is indicated in the quadrant. Representative original data are shown. C . Cells were treated with same as above and were analyzed for cell cycle by FACS as described in Materials and Methods. Experiments were done at least three times and representative original data are shown (* represents significant difference of p value of < 0.05 between JSI-124 treated and untreated cells and ** represents a p value of < 0.01).

Article Snippet: Rabbit polyclonal antibodies against phosphorylated or total c-Jun, JNK, p38, phosphor and total Erk1/2, XIAP and STAT3 antibodies, as well as siRNA for c-Jun (#6203) and Stat3 siRNA I (#6580) and control siRNA (#6201 fluorescein conjugate) were purchased from Cell Signaling Technology, VEGF antibody was purchased from ThermoScientific and Santa Cruz.

Techniques: Activation Assay, Western Blot, Staining, Control

Journal: BMC Cancer

Article Title: Cucurbitacin-I (JSI-124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B Leukemic Cells

doi: 10.1186/1471-2407-11-268

Figure Lengend Snippet: JSI-124 mediated c-Jun activation was not dependent on STAT3 expression in B-cell leukemia . A and B , Cells were transfected with siRNA-STAT or siRNA-c-Jun or siRNA non-target control. Then cells were treated with JSI-124 for additional 6 h. Cell extract was assayed for Western blotting for STAT3 and c-Jun. Knock-down of STAT3 or c-Jun was confirmed by western blotting with antibody to STAT3 or c-Jun. C , The same transfected cells were treated with JSI-124 for an additional 24 hours and then cells were analyzed for cell cycle analysis for G1 and G2 phase fractions in the cells transfected with siRNA-c-Jun or non-targeting control siRNA. Standard error was determined on the basis of three independent experiments and an asterisk represents significant difference (a p value of < 0.05) between JSI-124 treated and untreated controls. D , Cells were stimulated with JSI-124 and nuclear extracts incubated with the AP1 probe. Pulled down with streptavidin beads, the bound proteins were detected by SDS/polyacrylamide gel electrophoresis and western blotting. To control for specificity, a biotin-labeled scrambled probe was used along with no probe beads control. Blots were probed with antibody to c-Jun.

Article Snippet: Rabbit polyclonal antibodies against phosphorylated or total c-Jun, JNK, p38, phosphor and total Erk1/2, XIAP and STAT3 antibodies, as well as siRNA for c-Jun (#6203) and Stat3 siRNA I (#6580) and control siRNA (#6201 fluorescein conjugate) were purchased from Cell Signaling Technology, VEGF antibody was purchased from ThermoScientific and Santa Cruz.

Techniques: Activation Assay, Expressing, Transfection, Control, Western Blot, Knockdown, Cell Cycle Assay, Incubation, Polyacrylamide Gel Electrophoresis, Labeling

Journal: Nature Communications

Article Title: A local tumor microenvironment acquired super-enhancer induces an oncogenic driver in colorectal carcinoma

doi: 10.1038/s41467-022-33377-8

Figure Lengend Snippet: a ATAC-seq track at the PDZK1IP1 SE in primary CRCs from TCGA ( n = 81 independent tumors, merged into one track). b – d TRAP motif analysis at consensus open chromatin regions from the PDZK1IP1 SE in primary CRC, where sequences are compared against all human promoters with a Benjamini-Hochberg correction to generate a P -value. e Hallmarks of Cancer GSEA of RNA-seq expression data from PDZK1IP1 -high (top 50% mRNA expression) versus PDZK1IP1 -low (bottom 50% mRNA expression) primary CRCs from TCGA ( n = 342 independent tumors). f – i Hallmarks of Cancer GSEA enrichment signatures from PDZK1IP1 -high (top 50% mRNA expression) versus PDZK1IP1 -low (bottom 50% mRNA expression) primary CRCs from TCGA ( n = 342 independent tumors). j , k PDZK1IP1 expression levels by immunoblot in cytokine stimulated HT29 cells (10 ng/mL, 16 hours) or HT29 subcutaneous xenografts in nude mice. l H3K27ac ChIP-seq track of the PDZK1IP1 SE (underlined) in unstimulated or TNFα, IFNγ, and IL-6 co-stimulated HT29 cells at 10 ng/mL for 16 hours. Y-axes of all ChIP-seq tracks are scaled to the same range [0-122]. m Hallmarks of Cancer GSEA performed on differentially expressed genes from RNA-seq between HT29 xenograft tumors ( n = 3 independent tumors) and HT29 parental cells maintained in culture ( n = 3 biological replicates). n , o Immunoblot of PDZK1IP1 levels in HT29 xenografts in the presence of WT or deleted RELA or STAT3. p – s Immunoblot of PDZK1IP1 levels in HT29 cells with the indicated treatment for 16 hours. Source data are provided as a Source Data file. NES normalized enrichment score, FDR false discovery rate.

Article Snippet: The following primary antibodies were used for immunoblot: anti-PDZK1IP1 used 1:500 dilution (Sigma #HPA014907), anti-GAPDH used 1:10,000 dilution (Cell Signaling Technologies, CST #2118), anti-V5 used 1:1,000 dilution (CST #13202), anti-phosphorylated STAT3 at Y705 used 1:1,000 dilution (CST #9145), total STAT3 used 1:2,000 dilution (CST #9132), and NF-kB p65 encoded by RELA used 1:2,000 dilution (CST #8242).

Techniques: RNA Sequencing, Expressing, Western Blot, ChIP-sequencing

Journal: Genes & Development

Article Title: Fatty acid uptake activates an AXL–CAV1–β-catenin axis to drive melanoma progression

doi: 10.1101/gad.351985.124

Figure Lengend Snippet: Reagents used in this study

Article Snippet: Rabbit monoclonal anti-phospho-Stat3 (Tyr705; D3A7) , Cell Signaling Technology , 9145 , AB_2491009.

Techniques: Software, Transduction, Virus, Subcloning, Bacteria, Recombinant, Bradford Assay, Protease Inhibitor, RNA Extraction, Reporter Assay, Bicinchoninic Acid Protein Assay, Quantitative RT-PCR, Negative Control

Journal: Cancer Research

Article Title: Epidermal Growth Factor Receptor Cooperates with Signal Transducer and Activator of Transcription 3 to Induce Epithelial-Mesenchymal Transition in Cancer Cells via Up-regulation ofTWISTGene Expression

doi: 10.1158/0008-5472.can-07-0575

Figure Lengend Snippet: Figure 4. EGFR and STAT3 activate the human TWIST gene promoter. The human TWIST promoter–driven luciferase constructs were engineered to contain 824, 604, and 120 bp of the promoter and designated as phTWIST-824, phTWIST-604, and phTWIST-120, respectively. All transfection and determination of luciferase activity were carried out as previously described (24). Relative luciferase activity was derived from firefly luciferase activity after normalization against the activity of the transfection efficiency control, Recilla luciferase. All data represent the mean and SD from at least three independent experiments. A, EGFR activation by various ligands induces the TWIST gene promoter. MDA-MB-468 cells in six-well culture plates were transfected with phTWIST-824, phTWIST-604, and phTWIST-120. Recilla luciferase construct was cotransfected as transfection controls. After 24 h, transfected cells were serum-starved for 20 h and stimulated with 100 ng/mL EGF, TGF-a, and HB-EGF for 4 h. Harvested cells were lysed and subjected to luciferase assay. B, expression of EGFR and constitutive STAT3 (STAT3CA) activate the TWIST promoter. EGFR-null CHO-NEO cells (left) were cotransfected with phTWIST-120 and expression plasmids, pEGFR, pSTAT3CA, or combination. EGFR-negative NR-6 cells (middle), rat fibroblasts, were cotransfected with pEGFR and pSTAT3CA. After 48 h, transfected cells were lysed and luciferase activities determined. Right, CHO-EGFR cells were cotransfected with phTWIST-120 and indicated plasmids (pSTAT3CA and pSTAT3-DN). At 48 h, serum-starved cells were stimulated with EGF (100 ng/mL) for 4 h. Additionally, aliquots of CHO-EGFR cells were transfected with phTWIST-120, serum-starved, and pretreated with EGFR inhibitors (Iressa, 5 Amol/L; PD158780/PD, 10 Amol/L) and Jak/STAT3 inhibitor (AG490, 10 Amol/L) before 4 h of EGF stimulation. Control phTWIST-120 transfected cells were treated with EGF (Mock) for 4 h before analysis for luciferase activity. C, forced expression of dominant-negative STAT3 (STAT3-DN) and STAT3 small interfering RNA reduced TWISTexpression. Left, MDA-MB-468 cells were transfected with control and STAT3-DN vectors and, 48 h later, harvested and subjected to Western blot analysis for TWISTand a-tubulin expression. Right, Forced expression of STAT3 small interfering RNA reduced TWISTexpression. MDA-MB-468 cells were transfected with control small interfering RNA [nonspecific (N.S.), small interfering RNA, and STAT3 small interfering RNA]. After 48 h, transfected cells were harvested and subjected to Western blot analysis for STAT3, TWIST, and h-actin expression. D, involvement of nuclear EGFR in EGF-responsiveness of the human TWIST gene promoter. CHO-NEO, CHO-EGFR, CHO-EGFR-NLS cells were transfected with phTWIST-120, serum-starved at 24 h posttransfection, and treated with 100 ng/mL EGF and TGF-a for 4 h. Harvested cells were lysed and subjected to luciferase assay.

Article Snippet: Rabbit polyclonal phosphorylated STAT3 (p-STAT3; Y-705), phosphorylated EGFR (Y-1045), and Akt/phosphorylated Akt antibodies were from Cell Signaling.

Techniques: Luciferase, Construct, Transfection, Activity Assay, Derivative Assay, Control, Activation Assay, Expressing, Dominant Negative Mutation, Small Interfering RNA, Western Blot

Journal: Cancer Research

Article Title: Epidermal Growth Factor Receptor Cooperates with Signal Transducer and Activator of Transcription 3 to Induce Epithelial-Mesenchymal Transition in Cancer Cells via Up-regulation ofTWISTGene Expression

doi: 10.1158/0008-5472.can-07-0575

Figure Lengend Snippet: Figure 5. Identification of STAT3-targeted region within the human TWIST promoter. A, schematic illustration of the proximal region of the human TWIST promoter. The human TWIST proximal promoter contains two putative STAT3-binding elements. A TATAA box is located at nt 32 to 28, relative to the transcription start site. Site-directed mutagenesis was done to generate the phTWIST-120/MA mutant that contains multiple nucleotide substitutions (underlined) at nt 116 to 107 region and the phTWIST-120/MB mutant with nucleotide changes (underlined) at nt 99 to 96. The pTWIST-94 was additionally generated to remove the putative STAT3-binding sites and thus contains the minimal promoter up to 94 bp. B, mutation at the putative STAT3-binding site II significantly reduced the ability of the human TWIST promoter to respond to EGFR ligands. MDA-MB-468 cells were transfected with phTWIST-120, phTWIST-94, phTWIST-120/MA, and phTWIST-120/MB as previously described. After 48 h, serum-starved transfected cells were stimulated with EGF (100 ng/mL) for 4 h before determination of luciferase activities. All date represent the mean and SD from three independent experiments. C, biotinylated oligonucleotides precipitation assay. These studies were done to determine the degree of the binding of STAT3 to the TWIST promoter fragments. MDA-MB-468 cells untreated and treated with EGF (100 ng/mL) for 1 h were harvested, and nuclear lysates were extracted. Nuclear extracts were then subject to binding affinity evaluation to a number of biotinylated oligonucleotides, namely, STAT3-BS/ APRE (30, 31), TWIST-120/TWIST, TWIST-120/MA, and TWIST-120/MB. Biotinylated oligos were then precipitated by avidin beads, washed and subjected to Western blot analysis for p-STAT3 and EGFR. D, nuclear STAT3, but not nuclear EGFR, binds to the human TWIST gene promoter. A431 cells were serum-starved and stimulated without and with EGF (100 ng/mL) for 30 min and subjected to the in vivo binding assay ChIP, as we previously described. Briefly, EGFR monoclonal antibody (Neomarkers, Ab13) and STAT3 polyclonal antibody (Santa Cruz, C-20) were used in immunoprecipitation. IgG was used as negative control for immunoprecipitation, whereas input chromatins were used as positive controls for PCR and for equal loading. The c-fos promoter was also amplified as a positive control for both EGFR and STAT3 binding.

Article Snippet: Rabbit polyclonal phosphorylated STAT3 (p-STAT3; Y-705), phosphorylated EGFR (Y-1045), and Akt/phosphorylated Akt antibodies were from Cell Signaling.

Techniques: Binding Assay, Mutagenesis, Generated, Transfection, Luciferase, Avidin-Biotin Assay, Western Blot, In Vivo, Immunoprecipitation, Negative Control, Amplification, Positive Control