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1.
FIG. 4

FIG. 4. From: Engagement of the Cellular Receptor for Glycoprotein B of Human Cytomegalovirus Activates the Interferon-Responsive Pathway.

gB-mediated signaling is a direct response requiring transcription. Cycloheximide (CX; 100 μg/ml) was added to HF cells for 2 h before and during the 6-h stimulation period, while actinomycin D (ActD; 20 μg/ml) was present during the 6-h stimulation period. RNA was harvested at 6 h poststimulation and subjected to RT-PCR as for Fig. 1 and 3.

Kathleen A. Boyle, et al. Mol Cell Biol. 1999 May;19(5):3607-3613.
2.
FIG. 3

FIG. 3. From: Engagement of the Cellular Receptor for Glycoprotein B of Human Cytomegalovirus Activates the Interferon-Responsive Pathway.

Kinetic analysis of gene expression. HF cells were incubated with gB-S, HCMV, or IFN-α and harvested for mRNA-dependent RT-PCR analysis at increasing time intervals as indicated in minutes. PCR primer pairs for c-fos and ISG54 were used to assess upregulation of IFN-responsive genes; the β-actin gene was used as a housekeeping control gene.

Kathleen A. Boyle, et al. Mol Cell Biol. 1999 May;19(5):3607-3613.
3.
FIG. 7

FIG. 7. From: Engagement of the Cellular Receptor for Glycoprotein B of Human Cytomegalovirus Activates the Interferon-Responsive Pathway.

Incubation with gB stimulated the MAPK pathway. Fibroblasts were serum starved for 18 h prior to ligand stimulation. Cells were incubated in medium alone (M) or stimulated with FBS, IFN-γ (I-γ) or IFN-α (I-α) for 10 min. Cells were stimulated (Stim.) with gB or HCMV for 5-, 15-, or 30-min intervals. The cytoplasmic fraction was isolated and subjected to SDS-PAGE and immunoblotting. Reciprocol blots were probed with the anti-ACTIVE ERK1/2 polyclonal or pan ERK1/2 polyclonal antibody and detected with a secondary goat anti-rabbit HRP-conjugated antibody in conjunction with a chemiluminescent substrate.

Kathleen A. Boyle, et al. Mol Cell Biol. 1999 May;19(5):3607-3613.
4.
FIG. 5

FIG. 5. From: Engagement of the Cellular Receptor for Glycoprotein B of Human Cytomegalovirus Activates the Interferon-Responsive Pathway.

(A) HSPGs are not required for gB-mediated signaling. Fibroblasts were either mock treated (serum-free medium) or incubated with heparinase (2 U/ml) for 60 min at 37°C. The cells were washed, incubated with the signaling ligand for 60 min at 37°C, washed, and harvested for total RNA 6 h poststimulation. (B) Fibroblasts were incubated with IFN-α or IFN-γ for 30 min at 4°C prior to the addition of 35S-labeled gB. Homologous cold competition blocked gB binding by greater than 95% (not shown).

Kathleen A. Boyle, et al. Mol Cell Biol. 1999 May;19(5):3607-3613.
5.
FIG. 1

FIG. 1. From: Engagement of the Cellular Receptor for Glycoprotein B of Human Cytomegalovirus Activates the Interferon-Responsive Pathway.

gB-mediated upregulation of cellular mRNA. (A) HF cells were incubated with medium alone (mock), gB-S (500 μg/ml), IFN-α (1,000 U/ml), IFN-γ (100 U/ml), or HCMV (10 PFU/ml). After 6 h of stimulation, total cellular RNA was isolated and RT-PCR analysis was performed with oligo(dT)-primed RNA and three pairs of PCR primers (OAS, ISG54, β-actin). PCR-amplified products were separated on a 1% agarose gel by electrophoresis and visualized by ethidium bromide staining. (B) HF cells were incubated with increasing concentrations of gB-S (0.05 to 500 μg/ml) for 6 h and subjected to RT-PCR analysis as for panel A.

Kathleen A. Boyle, et al. Mol Cell Biol. 1999 May;19(5):3607-3613.
6.
FIG. 2

FIG. 2. From: Engagement of the Cellular Receptor for Glycoprotein B of Human Cytomegalovirus Activates the Interferon-Responsive Pathway.

gB-mediated signaling requires native structure. Prior to cell stimulation, gB (200 μg/ml) was denatured by heating to 70 or 94°C for 15 min, reduced by the presence of DTT (100 μg/ml) for 30 min at 37°C, or proteolytically digested with trypsin for 60 min at 37°C. As a control, cells were stimulated with BSA (500 μg/ml) under identical conditions. (B) The protein profiles for the heat-denatured and DTT-treated samples were determined by SDS-PAGE and immunoblotting with an antibody specific for an epitope on the carboxy-terminal domain. The positions of the dimeric (gB-D), monomeric (gB-M), carboxy-terminal (gB-C) fragments and the heat-induced aggregate (heat agg.) are indicated.

Kathleen A. Boyle, et al. Mol Cell Biol. 1999 May;19(5):3607-3613.
7.
FIG. 6

FIG. 6. From: Engagement of the Cellular Receptor for Glycoprotein B of Human Cytomegalovirus Activates the Interferon-Responsive Pathway.

Protein kinase activation is involved in gB signaling. (A) OAS and ISG54 mRNA levels were assessed in fibroblasts that were treated 2 h before and during stimulation with medium alone (Mo), ligand in the absence of inhibitor (None), solvent (Sol.), 50 nM calphostin C (CalC), 27.5 μM H7, or 29.5 μM H8. Quantitation of the relative yields of PCR product was determined with a Gel Documentation device (Bio-Rad). Extrapolation of the mean pixel density in each band to the relative percent volume allowed comparisons within an individual PCR. Each value was graphed as percent volume control relative to the untreated PCR product (None). (B) Fibroblasts were incubated with medium alone (Mo), ligand in absence of inhibitor (None), solvent (Sol.), 185 μM genistein (Gen), or 49.4 μM tyrphostin A25 (Tyr) 2 h before and during stimulation. RT-PCR analysis was performed to assess the effects of these inhibitors on cellular gene expression. Quantitation of the relative yields of the PCR products was determined as described for panel A. V, HCMV.

Kathleen A. Boyle, et al. Mol Cell Biol. 1999 May;19(5):3607-3613.

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