Visualizing PI(4)P Phosphorylation Kinetics

The kinetics of PI(4)P phosphorylation was measured on SLBs formed in ibidi chambers and visualized using TIRFM as previously described (Hansen et al., 2019 (link)). Reaction buffer contained 20 mM HEPES pH 7.0, 150 mM NaCl, 1 mM ATP, 5 mM MgCl₂, 0.5 mM EGTA, 20 mM glucose, 200 µg/ml β-casein (Thermo Fisher Scientific, #37528), 20 mM BME, 320 µg/ml glucose oxidase (Serva, #22780.01 Aspergillus niger), 50 µg/ml catalase (Sigma, #C40-100MG Bovine Liver) and 2 mM Trolox (UV treated; Hansen et al., 2019 (link)). Perishable reagents (i.e. glucose oxidase, catalase and Trolox) were added 5–10 min before image acquisition. For all experiments, we monitored the change in PI(4)P or PI(4,5)P₂ membrane density using solution concentrations of 20 nM Alexa647–DrrA(544-647) or 20 nM Alexa488–PLCδ1, respectively.