Journal: Journal of Biological Chemistry

Article Title: Modulation of T Cell Cytokine Production by Interferon Regulatory Factor-4

doi: 10.1074/jbc.m205895200

Figure Lengend Snippet: FIG. 1. Early activation events in IRF-4-transfected cells. A, whole cell extracts were prepared from Jurkat cells stably transfected with either a control or an IRF-4 expression vector, electrophoresed on a 7% SDS-polyacrylamide gel, and then analyzed by Western blotting using an anti-IRF-4 antibody (upper panel). The blot was later stripped and reprobed with a -actin antibody (lower panel) to ensure for equal loading. Extracts from untransfected Jurkat cells and HUT 78 served, respectively, as negative and positive controls. B, Jurkat-transfected cells were either left unstimulated or were stimulated with PMA (50 ng/ml) and ionomycin (1 M) for 24 h. The cells were then harvested and stained with either a phycoerythrin-labeled anti-CD69 (upper panel) or a phycoerythrin-labeled anti-CD25 antibody (lower panel) and analyzed by flow cytometry. Filled histograms represent unstimulated cells, whereas empty histograms represent cells stimulated with PMA and ionomycin. Left panel, vector transfectants; right panel, IRF-4 transfec- tants. Not shown is staining with an isotype-matched control, which did not reveal any significant differences between control and IRF-4 transfectants.

Article Snippet: Cell Lines and Cultures—The Jurkat (human T cell leukemia) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Activation Assay, Transfection, Stable Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Staining, Labeling, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: Modulation of T Cell Cytokine Production by Interferon Regulatory Factor-4

doi: 10.1074/jbc.m205895200

Figure Lengend Snippet: FIG. 4. IRF-4 transactivates the human IL-2 and IL-4 promoters. Control and IRF-4 Jurkat-transfected cells were transiently transfected with a luciferase reporter construct driven either by the human IL-2 promoter (left panel) or the human IL-4 promoter (right panel). The transfected cells were equally split into two 2-ml aliquots and then incubated for 4 h in the presence or absence of PMA (50 ng/ml) and ionomycin (1 M). The data are presented relative to the activity of the reporter construct in unstimulated control cells, which was set to 1.0, as indicated in each experiment. Results show the mean S.E. of five (for the IL-2 promoter) and six (for the IL-4 promoter) independent experiments.

Article Snippet: Cell Lines and Cultures—The Jurkat (human T cell leukemia) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Control, Transfection, Luciferase, Construct, Incubation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Modulation of T Cell Cytokine Production by Interferon Regulatory Factor-4

doi: 10.1074/jbc.m205895200

Figure Lengend Snippet: FIG. 6. IRF-4 can act as a transactivator of the P1-IRF element. Control and IRF-4 Jurkat cells were transfected with a luciferase re- porter construct driven by either an oligomerized P1-IRF wt or an oligomerized P1-IRFM3 element. The transfected cells were equally split into two 2-ml aliquots and then incubated for 4 h in the presence or absence of PMA (50 ng/ml) and ionomycin (1 M). The data are presented relative to the activity of the reporter construct in unstimu- lated control cells, which was set to 1.0, as indicated, in each experi- ment. Results show the mean S.E. of three independent experiments.

Article Snippet: Cell Lines and Cultures—The Jurkat (human T cell leukemia) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Control, Transfection, Luciferase, Construct, Incubation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Modulation of T Cell Cytokine Production by Interferon Regulatory Factor-4

doi: 10.1074/jbc.m205895200

Figure Lengend Snippet: FIG. 7. IRF-4 cooperates with NFAT in driving T cell cytokine production. A, vector and IRF-4 Jurkat cells were co- transfected with a luciferase reporter con- struct driven by the human IL-4 promoter and either an NFATc1 expression vector or equivalent amounts of an empty vector. The transfected cells were equally split into two 2-ml aliquots and then incubated for 4 h in the presence or absence of PMA (50 ng/ml) and ionomycin (1 M). The data are presented relative to the activity of the reporter construct in vector control cells, which was set to 1.0, as indicated, in each experiment. Results show the mean S.E. of four independent experi- ments. B, control and IRF-4-transfected cells were either left unstimulated or stimulated with PMA and ionomycin as indicated in the legend to Fig. 2. Stimula- tions were conducted in the presence or absence of cyclosporin A (1 g/ml) or FK506 (10 ng/ml) as indicated. Superna- tants were then collected and analyzed for their cytokine content by ELISA. Data shown are representative of four inde- pendent experiments and performed on three independent sets of transfectants.

Article Snippet: Cell Lines and Cultures—The Jurkat (human T cell leukemia) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Plasmid Preparation, Transfection, Luciferase, Expressing, Incubation, Activity Assay, Construct, Control, Enzyme-linked Immunosorbent Assay

Journal: Developmental biology

Article Title: Fertilization stimulates 8-hydroxy-2'-deoxyguanosine repair and antioxidant activity to prevent mutagenesis in the embryo.

doi: 10.1016/j.ydbio.2015.07.024

Figure Lengend Snippet: Fig. 1. OGG1 is under-represented in MII-stage mouse oocytes. (A) Quantitative PCR analysis revealed a low abundance of OGG1 and APE1 mRNA within the murine oocyte (relative to B2M and Gusβ), whilst XRCC1 transcripts were present at significantly higher levels. (B) Immunoblotting analysis of the protein composition of these oocytes identified bands corresponding to APE1 and XRCC1 at 35 kDa and 85 kDa respectively; whilst a 37 kDa band corresponding to OGG1 could not be detected at this con- centration of protein (100 oocytes per lane, equivalent to 1 mg protein). Positive controls; rOGG1, rAPE1 and Jurkat cell lysate respectively (1 mg). (C, D) In support of Western blotting data, immunocytochemistry identified fluorescence associated with APE1 and XRCC1 throughout the ooplasm. Fluorescence relating to OGG1 was low, however upon pixel intensity analysis was still found to be significantly greater than the ‘secondary only’ control (“C”). Scale bar¼50 mm. Mean7SEM values are plotted in histograms. Independent replicates were conducted with a minimum of 40 oocytes per replicate. *Po0.05, **Po0.01, ***Po0.001.

Article Snippet: 1 mg of recombinant OGG1 (rOGG1), recombinant APE1 (rAPE1) (both from New England Biolabs, Ipswich, MA, USA), and Jurkat whole cell lysate (Novus Biologicals, Littleton, CO, USA) was loaded onto the polyacrylamide gels as a positive control for OGG1, APE1 and XRCC1 antibodies respectively.

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

Journal: Biology of reproduction

Article Title: Transcriptional regulation of cyclooxygenase-2 gene in ovine large luteal cells.

doi: 10.1095/biolreprod65.5.1565

Figure Lengend Snippet: FIG. 8. Western Blotting of USF-1 and USF-2 proteins in nuclear extracts from ovine large luteal cells untreated (control) or treated with PGF2a (100 nM) for 1 h. The blot was analyzed with anti-USF-1 antibody (A) or anti- USF-2 antibody (B). Whole cell lysates from Jurkat cells are used as a positive control for USF-2. Molecular masses of USF-1 (43 kDa) and USF- 2 (44 kDa) are indicated with an arrow.

Article Snippet: Polyclonal antibodies against upstream stimulatory factor (USF)-1 and USF-2, donkey anti-rabbit IgGhorseradish peroxidase (HRP), and Jurkat whole cell lysates were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Control, Positive Control

Journal: International journal of molecular medicine

Article Title: Expression of A disintegrin and metalloprotease 10 in pancreatic carcinoma.

doi: 10.3892/ijmm_00000463

Figure Lengend Snippet: Figure 1. Expression of ADAM10 in pancreatic cancer cell lines and pancreatic tissue samples. A. Expression of ADAM10 protein in pancreatic cancer cells, 6 of 7 tested cell lines displayed protein expression for ADAM10. Jurkat cells served as a positive control. B. Expression of ADAM10 mRNA in pancreatic cancer (PDAC) cells. All tested cell lines displayed ADAM10 mRNA expression. C. Expression of ADAM10 mRNA in pancreatic tissues (qRT-PCR). ADAM10 mRNA expression levels in normal pancreas NP) (n=25), chronic pancreatitis (CP) (n=55) and pancreatic cance (PDAC) tissue (n=79). D. Expression of ADAM10 in pancreatic tissues. ADAM10 protein expression in NP (n=2), CP (n=2) and PDAC (n=7) tissue. Jurkat cells served as positive control. In contrast with the results of the qRT-PCR, there was a trend toward upregulation of ADAM10 protein in CP as well as in PDAC compared to NP. E. Expression and localization of ADAM10 in pancreatic tissues based on immunohistochemistry. Representative ADAM10 immunoreactions in NP, CP and PDAC are shown. The lack of staining in consecutive negative control tissue sections is shown as inserts.

Article Snippet: Protein lysates of Jurkat cells served as positive control for the ADAM10 antibody and ZR-75-1 cell lysate (Santa Cruz, Heidelberg, Germany) for E-cadherin antibodies.

Techniques: Expressing, Positive Control, Quantitative RT-PCR, Immunohistochemistry, Staining, Negative Control