Fig. 3. Activation of Dd-STATc by heat shock and ATP depletion. (A) Ax2 cells were allowed to develop for four hours in shaken suspension at 21°C and then transferred to a heating block at 33°C. At the indicated times thereafter, aliquots were harvested and the specific tyrosine phosphorylation level of Dd-STATc was determined by western transfer. As a positive control for the induction, a portion of the cells was maintained at 21°C, DIF was added to a final concentration of 100 nM and an aliquot was analysed in parallel with the heat shock samples. The results are therefore quantitatively comparable and heat shock appears to be a more effective activator than DIF. (B) Ax2 cells were allowed to develop for 4 hours in shaken suspension and di-nitro phenol (DNP) was added to a final concentration of 50 µM. At the indicated times thereafter, aliquots were harvested and the specific tyrosine phosphorylation level of Dd-STATc was determined by western transfer. DIF was added to a final concentration of 100 nM and an aliquot was analysed in parallel with the oxidative shock samples. The results are therefore quantitatively comparable and oxidative shock is a much less effective inducer than DIF. (N.B. This experiment and the experiment described above, in Fig. 3A, are not directly comparable because they were performed at different times.)