FIGURE 7:

In vitro tyrosine phosphorylation of STATc by Pyk2 and Pyk3 and the role of intracellular cGMP. (A) Tyrosine phosphorylation of STATc using immunopurified kinases. Ax2 cells transformed with myc-Pyk2 or myc-Pyk3, at 4 h of suspension development, were treated with sorbitol for 5 and 15 min or 8Br-cGMP (a membrane-permeable cGMP analogue at 20 mM) for 5 min, then lysed, and the enzyme immunopurified from them was assayed for STATc tyrosine kinase activity with GST-STATcΔ as substrate in a phospho-STATc Western assay. (B) 8Br-cGMP induced activation of STATc in single- and double-null cells Tyrosine phosphorylation of STATc was assayed after 8Br-cGMP treatment at 20 mM for 5 min. The blot was reprobed with total STATc antibody 7H3. (C) 8Br-cGMP induced nuclear enrichment of STATc in single- and double-null cells. Nuclear localization of STATc after 8Br-cGMP treatment for 5 min was analyzed by immunofluorescence staining with total STATc antibody. Bar, 10 μm.