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Fig. 7. BNIP-H alters the steady-state levels of glutamate and inhibits glutaminase activity. (A) 293T cells were transfected with the indicated plasmids and harvested after 24 hours. Glutamate amounts in the cell lysate (black bars) and in the medium (grey bars) were determined enzymatically as described in the Materials and Methods. The results presented are means ± s.d. of three independent transfection studies, with the measurements done in triplicate. In each experiment, KGA amounts were detected by western blotting to ensure equal expression in double-transfected cells. A representative expression analysis is shown at the bottom; signals for anti-ß-tubulin demonstrate equal loading of the whole-cell lysates. EF1A1 was used as a control and had no significant effect on the endogenous glutamate level in the cell (P=0.10) and in the medium (P=0.89). White bars show the total amount of glutamate in both the lysate and medium. Capital letters (A,B), small letters (a,b) and symbols (
, ß) indicate values for intracellular, medium and total glutamate amounts, respectively. Difference between values not sharing the same letters or symbols are statistically significant at P<0.05 by analysis of variance and the Newman-Keuls multiple range test (StatsDirect). Bars showing obvious similarities in values are excluded from labelling for clarity purposes. (B) Lysate from 293T cells over-expressing FLAG-tagged KGA was incubated with equal amounts (5 µg) of bacterially expressed purified GST-tagged BNIP-H FL, GST-BNIP-H (aa 1-190) or GST. After pre-incubation, glutamine was added to the samples for 0, 2, 5 and 10 minutes. Samples were then precipitated and total amounts of glutamate were determined as in A. Results are means ± s.d. of three independent enzyme assays
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