A GST pulldown assay was performed as described previously [13]. These supports were used with whole cell lysates typically prepared from 10 7 lymphoblastoid cells. Immunostaining and digital image capture were performed as described elsewhere.
Applied Biosystems systems software was used for analysis. Ct values were determined for the internal control TBP and for the test genes at the same threshold level in the exponential phase of the PCR curves.
Dissociation curve analysis was performed after every run to check the specificity of the reaction. Three or four reactions each in triplicate were run for each gene and the standard deviation was calculated. To measure lactate concentration, the cell culture medium was collected. Cells were treated with 1 mM NiCl 2 , mimicking hypoxia-like conditions.
Proteins from whole lymphoblastoid cell lysates were captured by the support and then analyzed by Western blotting. A — GST pulldown assay and Western blotting from lymphoblastoid cell lysates. B — Immunoprecipitation and Western blotting from lymphoblastoid cell lysates. A — Western blotting following the GST pulldown assay from lymphoblastoid cell lysates. Conversely, the HIF1A signal green was observed mainly in the cytoplasm of mitogen-activated B-cells, while in LCL it showed both a cytoplasmic and a nuclear distribution.
A — Western blotting of cellular subfractions. The membrane was probed with mouse anti-HIF1A and anti-actin antibodies left panel. Nuclear DNA is stained in blue. Given that EBV-infected immunoblasts are not normally exposed to hypoxia either in vitro or in their natural niche in vivo , and given the involvement of HIF1A in glycolysis, we examined the expression of several genes of the glycolytic pathway Figure 5.
Untreated and cells treated with NiCl 2 were compared. The concentrations of lactate and pyruvate were 5- and 3-fold higher in the LCLs than in mitogen-activated B-cells.
EBV-infected B-cells showed intermediate values for lactate, pyruvate and lactate dehydrogenase Figure 6A.
Because enhanced ATP synthesis leads to elevated levels of reactive oxygen species ROS , their levels were assayed in all three groups of cells. Freshly EBV-infected cells showed intermediate values. It is powerfully induced by hypoxia at both mRNA and protein levels [14].
Under normal conditions, the HIF1A protein level is low because of proteasome-dependent degradation. At high oxygen levels, a few prolines e. Our findings show that HIF1A is stabilized in EBV-transformed B-cells and, given that mRNA levels are not changed, it seems likely that the stabilization is due to the inhibition of hydroxylation, as we have demonstrated here. PDK1 phosphorylates the mitochondrial pyruvate dehydrogenase complex responsible for oxidative decarboxylation of pyruvate.
The level of GLUT1, a glucose transporter responsible for glucose uptake, is high in thyroid [19] and colorectal cancers [20]. PGK1, a phosphoglycerate kinase, is secreted by fibrosarcoma cells at levels several-fold higher than for normal fibroblasts [21]. Latency, on the other hand, has only been detected in situ in latently infected humans. It is characterized by the presence of very low numbers of viral episomes that appear to express the RNA for only one protein Latent Membrane Protein 2 in B cells that bear the markers of a non-activated resting memory subset.
Lymphoblastoid marmoset cell line B Callithrix genus was established by the infection of marmoset B cells with EBV isolated from a patient with infectious mononucleosis. B cell line provides a source of EBV to generate continuous B lymphoblastoid lines from human donors. By DNA profiling, it has been confirmed that B cell line was actually derived from a cotton-top tamarin Saguinas oedipus instead of marmoset. B cell immortalization with Epstein-Barr virus. Chan School of Public Health, was able to track the long-term health of about 10 million active duty military members, thanks to a year collaboration with the U.
At the start of their service, these members have their blood screened for HIV and are screened regularly every two years afterward. This meant the researchers could test those same blood samples for EBV as well. Over the year study period, individuals were diagnosed with multiple sclerosis during their service. All but one of those people contracted the virus before their eventual MS diagnosis. By contrast, people in the control group who never developed MS were also less likely to have caught EBV during the study period.
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