Agonist-driven ROS production in primed human neutrophils involves sequential activation of 2 PI3K subtypes. This figure depicts bacteria (green rectangles) being recognized by the cell surface fMLP receptor, triggering phagocytosis and activation of the NADPH oxidase complex. Within 10 seconds of fMLP binding, PIP₃ levels rise via the G-protein–dependent activation of class IB PI3K (p110γ). A second peak of PIP₃ is observed around 1 to 2 minutes, coinciding with the peak levels of superoxide production. This second peak is largely dependent on class IA PI3K (thick arrow), but absolutely requires prior activation of the class IB PI3K. The steps leading from the initial PIP₃ generation to class IA PI3K activation are not established (dashed lines). However, the Src family kinase inhibitor PP1 attenuates the second peak of PIP3, implicating this kinase family in the pathway. PIP₃ and other PI3K products promote NADPH oxidase function.