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. 2021 Feb 22;118(9):e2025343118. doi: 10.1073/pnas.2025343118

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Fig. 6. — Dissection of phosphoinositide restoration after depletion. (A) Averaged time course of heterologous nFRET_eff between PH_PLCδ1 and L10 or S15 measuring PtdIns(4,5)P₂ recovery in L_o and L_d domains after brief VSP stimulation. A 2-s voltage pulse to 100 mV stimulated VSP to dephosphorylate PtdIns(4,5)P₂ to PtdIns4P. The traces in the dashed box expand the first 20 s of recovery after the end of VSP stimulation (L10: n = 11 cells and S15: n = 9 cells). The absolute resting FRET_eff used for normalizations was L10: 23.1 ± 4.4% and S15: 21.6 ± 3.8%. (B and C) Paired-pulse stimulation of VSP before and after 3-min treatment with MβCD to measure the sensitivity to change in domain sizes for L_o (B) and L_d (C) (L10: n = 7 cells and S15: n = 6 cells). (D) Summary of PtdIns(4,5)P₂ recovery time constants after VSP stimulation. (E) Time course of PtdIns4P recovery after 20-s stimulation of the M₁R receptor (L10: n = 9 cells and S15: n = 9 cells). The absolute resting FRET_eff used for normalizations was L10: 10.9 ± 2.5% and S15: 12.4 ± 1.5%. (F and G) The same when MβCD was applied immediately after (F) (L10: n = 12 cells and S15: n = 13 cells) or 3 min before (G) (L10: n = 10 cells and S15: n = 9 cells) the Oxo-M. The absolute resting FRET_eff used for normalizations was for F, L10: 9.6 ± 1.4% and S15: 9.4 ± 1.4%, and, for G, L10: 11.0 ± 1.4% and S15: 12.8 ± 1.8%. (H) PtdIns4P recovery time constants in L_o and L_d domains after receptor activation. *P < 0.05, **P < 0.005, ***P < 0.001, n.s., not significant.