(A–B) Examination of the PI(4,5)P2-dependent behavior of PM-associated GRAMD2a-eGFP (A) and GRAMD1a-eGFP (B). PM PI(4,5)P2 was reversibly depleted by 10 s addition (middle panel) and removal (left panel) of 10 μM of the muscarinic agonist oxotremorineM (OxoM) as monitored by the PI(4,5)P2 marker CFP-PH-PLCδ1. Normalized intensity of fluorescent proteins used in PI(4,5)P2 depletion experiments shown in lower panels of (A and B). Representative images shown from at least 12 cells that were obtained from two biological replicates. Fluorescence intensity dynamics were analyzed for all cells. (C) Western Blot analysis of centrifugation-based liposome binding assays with recombinant His6-GRAMD2aΔTM and liposomes of different composition. Control PM-like liposomes: 85% PC: 15% PS; PM-like liposomes with PI: 85% PC: 10% PS: 5% PI or 85% PC: 15% PI; PM-like liposomes with PI(4)P: 85% PC: 14% PS: 1% PI(4)P, 85% PC: 10% PS: 5% PI(4)P, or 85% PC: 15% PI(4)P; PM-like liposomes with PI(4,5)P2: 85% PC: 14% PS: 1% PI(4,5)P2, 85% PC: 10% PS: 5% PI(4,5)P2, or 85% PC: 15% PI(4,5)P2. S and P indicate supernatant and pellet, respectively. (D) Quantification of liposome binding experiments, n = 4 (biological replicates), Standard Error shown.