Figure 4. Membrane-anchored phosphorylated (pY) peptides synergistically enhance Dy647-PI3Kβ membrane binding in the presence of Rac1(GTP).

(A) Cartoon schematic showing membrane conjugation of Cy3-Rac1 and AF488-pY on membranes containing unlabeled Rac1 and pY. (B) Representative TIRF-M images showing localization of Cy3-Rac1 (1:10,000 dilution) and AF488-pY (1:30,000 dilution) after membrane conjugation in the presence of 30 µM Rac1 and 10 µM pY. Membrane surface density equals ~4000 Rac1/µm² and ~5000 pY/µm².(C) Representative TIRF-M images showing the equilibrium membrane localization of 5 pM and 10 nM Dy647-PI3Kβ measured in the presence of membranes containing either pY/Rac1(GDP) or pY/Rac1(GTP). (D ) Bulk membrane recruitment dynamics of 10 nM Dy647-PI3Kβ measured in the presence of pY/Rac1(GDP) or pY/Rac1(GTP). (E) Single molecule dwell time distributions measured in the presence of 5 pM Dy647-PI3Kβ on supported membranes containing pY/Rac1(GDP) or pY/Rac1(GTP). (F) Step size distributions showing single molecule displacements from >500 Dy647-PI3Kβ particles (>10,000 steps) in the presence of either pY/Rac1(GDP) or pY/Rac1(GTP). Membrane composition: 96% DOPC, 2% PI(4,5)P₂, 2% MCC-PE.