Fig. 3. Tor^GOF activates and associates with Mrl. (A) Mrl activity was measured by a gel mobility shift assay in S2 cell extracts using an oligonucleotide (top strand sequence: GGATTTTTCCCGGAAATG. Bottom strand sequence: GACCATTTCCGGGAAAAA) optimal for Mrl binding (Yan et al., 1996). Control (lane 1) shows basal levels of Mrl activity in S2 cells. Transfection of Hop (lane 4; 10 µg) resulted in a significant increase in Mrl DNA-binding activity. Transfection of DNA encoding wild-type Tor (lane 2; 10 µg) or Tor^GOF (lane 3; 10 µg) significantly increased the DNA-binding activity of Mrl to levels similar to those observed following Hop transfection. Cells treated with the vanadate-H₂O₂ (100 µM sodium orthovanadate, 1 mM hydrogen peroxide) (see Sweitzer et al., 1995) strongly activate Mrl (lane 5) and result in similar gel-shift bands. Addition of an anti-Mrl antibody caused a supershift of the protein-DNA complex (lane 6), suggesting it is due to Mrl-oligonucleotide association. (B) Tor protein was precipitated with anti-Tor antibody (Cleghon et al., 1996) from wild-type and tor^GOF embryo extracts, respectively. Note Mrl (80 kDa) was co-precipitated with Tor (135 kDa) from both wild-type and tor^GOF embryos in the presence of vanadate.

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