puma creepers
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PUMA is known to localize on the mitochondrial puma creepers membranes to initiate apoptosis upon appropriate stress [ 40 ; 41 ]. Rationalized by our data showing that EGFR and EGFRvIII co-express and interact with PUMA and that these two pathways are inversely linked to apoptosis, we examined whether EGFR/EGFRvIII might regulate PUMA sub-cellular localization. Thus, we analyzed the cytoplasmic/mitochondrial distribution of PUMA in the isogenic pair, U87MG-EGFRvIII and U87MG-vector cells.

The extent of PUMA mitochondrial translocalization, mtPUMA Index, is computed as described earlier in Materials and Methods. (A) PUMA is primarily localized in the cytoplasm of U87MG-EGFRvIII cells. U87MG-EGFRvIII cells treated puma fenty with vehicle control, staurosporin (ST, 1 uM) or anisomycin (AN, 100 ng/ml) were harvested and fractionated into mitochondrial and non-mitochondrial fractions. Protein extracts from both fractions were subjected to western blotting to detect PUMA.

PUMA puma slides is primarily localized in the cytoplasm of these cells, under unstressed and stressed conditions, as indicated by the low mtPUMA indices.Similar observations were further found in T98G GBM cells that naturally express EGFR ( Fig. 5c ). The modest detection of mitochondrial PUMA in these cells may potentially be the result of insufficient cytoplasmic EGFR to interact with and sequester all the PUMA molecules in the puma rihanna cytoplasm.

Importantly, siRNA-mediated EGFR expression knockdown led to a significant increase (16-fold) of mitochondrial PUMA ( Fig. 5c-left ), further suggesting the ability of EGFR to modulate PUMA mitochondrial translocalization. EGFR-specific siRNA was effective in reducing EGFR expression as shown by the western blots ( Fig. 5c-right ). In addition to GBM cells, we found PUMA to be localized in the cytoplasm of MDA-MB-468 human breast cancer cells .

That are known to express significant levels of endogenous EGFR [ 34 ]. Collectively, these results demonstrate that PUMA is sequestered in the cytoplasm of EGFR- and EGFRvIII-expressing GBM and breast cancer puma suede classic cells, constitutively and under apoptotic stress.To gain insight into the factors that modulate the interaction of EGFR with PUMA, we examined the requirement of EGFR activation for the EGFR-PUMA interaction.

U87MG-EGFR cells were serum-starved for 24 hrs and stimulated with EGF for 20 mins. The cells were harvested and subjected to immunoprecipitation/western blotting (left panel) and western blotting (right panel). An EGFR antibody was used to immunoprecipitate EGFR. Control IgG did not yield signals indicating specificity. Auto-phosphorylated Y1068 residue serves as an indicator for EGF-induced EGFR activation. (B) EGFR kinase [Bild: puma suede classic-642law.jpg] activity is not required for their interaction with PUMA.
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