Abstract

We have studied the dynamics of nuclear translocation during nuclear factor kappaB activation by using a p65(RELA)-enhanced green fluorescent protein (EGFP) fusion construct. Quantitation of expression levels indicates that EGFPRELA can be detected at physiological concentrations of about 60,000 molecules per cell. Stimulation of transfected fibroblasts with interleukin (IL)-1beta caused nuclear translocation of EGFPRELA, typically resulting in a 30-fold increase in nuclear protein at maximum induction and a concomitant 20% decrease in cytoplasmic levels. The response of individual cells to IL-1beta was graded, and the kinetics of nuclear translocation were dependent on the dose of IL-1beta and the level of EGFPRELA expression. The rate of nuclear uptake was saturable, and the time lag for uptake increased at higher EGFPRELA expression levels. Furthermore, nuclear translocation was reduced at less than saturating doses of IL-1beta suggesting that the pathway is limited by incoming signals. The response to IL-1beta was biphasic, demonstrating a decline in nuclear import rate at expression levels above three to four times endogenous. This correlated with the anti-apoptotic function of EGFPRELA which was more prominent at low expression levels and demonstrated successively less protection at higher levels. In comparison, transfection of p50 had no effect on the level of apoptosis and demonstrated some toxicity in combination with EGFPRELA.

Highlights

  • We have studied the dynamics of nuclear translocation during nuclear factor ␬B activation by using a p65(RELA)-enhanced green fluorescent protein (EGFP) fusion construct

  • To verify that EGFPRELA accurately reflects the nuclear translocation of endogenous RELA, transfected cells were treated with varying doses of IL-1␤ for 30 min, and the distribution of the fusion protein was analyzed (Fig. 4)

  • We find that either the protein is concentrated in the cytoplasm but impaired for nuclear translocation or it is aberrantly concentrated in the nucleus

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Summary

Introduction

We have studied the dynamics of nuclear translocation during nuclear factor ␬B activation by using a p65(RELA)-enhanced green fluorescent protein (EGFP) fusion construct. Calibrating the system using fibroblasts transfected with EGFP and comparison of cell extracts with purified EGFP protein by Western blot (Fig. 2B) indicates that relative fluorescence 1 corresponds to expression of approximately 500,000 molecules per cell.

Results
Conclusion

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