Percent changes in band intensities revealed the relative expression levels of the two ZAP-70 proteins. First, we note that the non-stimulated cells feature constitutive hyperphosphorylation of Y493. This is in line with previous reports, showing that phosphorylation of Y493 is a positive regulatory mechanism required for activation of ZAP-70 and lymphocyte antigen receptor function. Anti-CD3 stimulation slightly increased the Y493 phosphorylation compared to the basal level while Y492 phosphorylation remained unchanged. H2O2 stimulation alone caused a slight decrease of the Y493 phosphorylation, an effect reinforced in presence of anti-CD3 that also boosted Y492 phosphorylation. In contrast, stimulation with Na2VO4 alone or in combination with anti-CD3 caused a pronounced accumulation of phosphorylation at Y492, induced by the phosphatase inhibition, while the phosphorylation at Y493 remained indistinguishable from non-stimulated or anti-CD3stimulated cells.