Fig. 4. Interaction of PTP3 with STATc revealed by substrate-trapping. (A) In vitro binding of PTP3. STATc parental Ax2 cells and STATc-null cells were starved for 4 hours and then left untreated (control), exposed to DIF-1 (100 nM) or exposed to sorbitol (200 mM). After 5 minutes of treatment, extracts were prepared from the cells and subjected to affinity chromatography using the C to S substrate-trapping form of PTP3. The eluates from the columns were subjected to western transfer using a general phosphotyrosine-specific antibody (upper panel) or a tyrosine phosphorylation-specific STATc antibody (lower panel). The strongest DIF-1 and stress-induced signal detected by the phosphotyrosine-specific antibody is a 130 kDa species that corresponds to a previously described protein (Gamper et al., 1999). This protein co-migrates with the strongest signal detected by the STATc antibody and both species are absent from the STATc null. (B) In vivo interaction of PTP3 and STATc parental Ax2 cells or cells expressing the myc tagged C to S substrate-trapping form of PTP3 (myc:PTP3CS, labelled as PTP3CS) were starved for 4 hours and then left untreated (control), exposed to DIF-1 at 100 nM or exposed to sorbitol at 200 mM. After 5 minutes of treatment, extracts were prepared from the cells and immunoprecipitated using a myc antibody. After western transfer, the blot was stained using a general STATc antibody and a tyrosine phosphorylation-specific STATc antibody. We estimate that 5% of STATc protein in the cell is recovered by the co-IP procedure.