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1.
Figure 5

Figure 5. From: The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor– and MAP kinase–dependent interregulation.

Functional Ras is required for basal and trefoil-dependent pS2 transcription. AGS cells were cotransfected with a human gastrin promoter–luciferase reporter plasmid (gas-luc) or the pS2 promoter luciferase construct with or without dominant-negative Ras (N17Ras). To confirm the effect of N17Ras, the effect of EGF (10 ng/mL) on Ras-dependent () activation was assessed.

Douglas Taupin, et al. J Clin Invest. 1999 May 1;103(9):R31-R38.
2.
Figure 2

Figure 2. From: The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor– and MAP kinase–dependent interregulation.

Expression of trefoil peptide–encoding mRNA in gastric cells after stimulation with trefoil peptides. KATO-III cells were grown to confluence, serum-deprived (0.5% serum), and then stimulated for the indicated times with recombinant ITF (100 μg/mL), SP (100 μg/mL), or EGF (100 ng/mL) in the absence or presence of cycloheximide (25 μg/mL). Northern blots were performed with [α-32P]dCTP–labeled cDNA probes as described in Methods. CHX, cycloheximide.

Douglas Taupin, et al. J Clin Invest. 1999 May 1;103(9):R31-R38.
3.
Figure 1

Figure 1. From: The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor– and MAP kinase–dependent interregulation.

Gastric trefoil peptide RNA and protein is reduced in ITF–/– compared with wild-type (Sv129/C57Bl/6) mice. (a) Northern blots of mouse gastric antrum. Total RNA was hybridized with an [α-32P]dCTP–labeled rSP cDNA probe (), mouse ITF cDNA probe (), mouse-specific pS2 probe (), or a probe for the constitutive GAPDH transcript as described in Methods. (b) Western blot of gastric mucosal lysates resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane, incubated with rSP antibody (), and viewed by enhanced chemiluminescence.

Douglas Taupin, et al. J Clin Invest. 1999 May 1;103(9):R31-R38.
4.
Figure 3

Figure 3. From: The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor– and MAP kinase–dependent interregulation.

Effect of recombinant trefoil peptides on expression of human pS2 and SP reporters in transient transfection. (a) hSP (820 bp) and hpS2 (1,097 bp) promoters were cloned as detailed in Methods and subcloned into the luciferase reporter vector pGL2. (b) AGS cells were transfected with hSP-luc (left panels) or hpS2-luc (right panels) reporter constructs, followed by stimulation with human SP (top panels) or human ITF (bottom panels) peptides at the indicated final concentrations for 24 hours, as described in Methods. Results shown (mean ± SE) are representative of 3 independent experiments performed in triplicate.

Douglas Taupin, et al. J Clin Invest. 1999 May 1;103(9):R31-R38.
5.
Figure 7

Figure 7. From: The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor– and MAP kinase–dependent interregulation.

ITF binding to cells under nonpermissive conditions colocalizes with the transferrin receptor, but not with the EGF-R. AGS cells (pictured) or HT-29 cells were grown on glass, cooled to 4°C, and an excess (10 μg/mL) of ITF or ITF-thioredoxin was added. Incubations with primary antibodies were as follows. Top left: monoclonal anti-thioredoxin; top center: rabbit anti–EGF-R; top right: merged image showing minimal colocalization of ITF (red) and EGF-R (green). Similar staining was seen with anti-ITF staining in combination with monoclonal anti–EGF-R. Original magnification: ×200. Bottom left: rabbit anti-ITF; bottom center: clathrin-coated pits seen with monoclonal antibody to the transferrin receptor CD71; bottom right: merged image showing predominantly colocalization of ITF staining with CD-71. Original magnification: ×400.

Douglas Taupin, et al. J Clin Invest. 1999 May 1;103(9):R31-R38.
6.
Figure 6

Figure 6. From: The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor– and MAP kinase–dependent interregulation.

EGF-R–mediated stimulation of pS2 transcription. (a) Phosphorylation of EGF-R in AGS cells by ITF. Confluent, serum-starved AGS cells were stimulated with ITF (10 ng/μL) for the indicated times in minutes or with EGF for 5 minutes before cell lysis. Proteins were immunoprecipitated with anti–EGF-R, separated on SDS-PAGE, transferred to PVDF membranes, and blotted with the monoclonal anti-phosphotyrosine antibodies 4G10/PY20. (b) Transcriptional regulation of pS2 by trefoil peptides is mediated by EGF-R. AGS cells were transfected with the pS2-luciferase reporter construct in the presence or absence of a dominant-negative EGF-R (pHER653) expression construct, then stimulated 12 hours later with EGF or ITF. Relative light units (RLU) corrected for transfection efficiency were assessed by cotransfection with 1 μg pRL-TK (Renilla) luciferase.

Douglas Taupin, et al. J Clin Invest. 1999 May 1;103(9):R31-R38.
7.
Figure 4

Figure 4. From: The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor– and MAP kinase–dependent interregulation.

(a) MAP kinase activation by trefoil peptides. Confluent, serum-deprived KATO-III cells (top) or serum-starved AGS cells (bottom) were stimulated for the indicated times with human ITF or SP at 10 ng/μL, human EGF at 100 ng/mL, or genistein 50 μM (lane 5G). Cells were harvested by in-plate lysis in the presence of protease inhibitors, and 500 μg protein was immunoprecipitated with polyclonal anti-ERK1. Immunoprecipitates were incubated with myelin basic protein as substrate in the presence of [γ-32P]dATP and resolved on SDS-PAGE. (b) Lack of Jun kinase activation by addition of hSP to KATO-III cells. Cell lysates as above were immunoprecipitated with monoclonal anti–c-Jun. Kinase reactions were performed with c-Jun as substrate. (c) MAP kinase activation is necessary for cross-regulation of trefoil peptide transcription. AGS cells were transiently transfected with pS2-luc and/or cotransfected with an expression construct encoding the ERK-dephosphorylase PAC-1 (PMT2T-PAC1). Twelve hours after transfection, cells were stimulated with ITF (10 ng/μL) and/or the MEK-1 inhibitor PD098059 (25 μM).

Douglas Taupin, et al. J Clin Invest. 1999 May 1;103(9):R31-R38.

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